Method for enhancing ToF-SIMS detection sensitivity in single cell

Abstract

The invention relates to the field of biomedical engineering. The invention discloses a method for enhancing ToF-SIMS detection sensitivity in a single cell. The method comprises the following steps: S1, carrying out ITO glass surface treatment, preparing a nano capillary tube, and representing the nano capillary tube; s2, inoculating the MCF-7 cells to ITO glass, and culturing the MCF-7 cells; s3, carrying out treatment on the MCF-7 cells; s4, injecting the quantum dot solution into the MCF-7 cells under the action of voltage; s5, cleaning and freezing the ITO glass; and (S6) analyzing the MCF-7 cells by using ToF-SIMS (Tooth of Free- The solution at the tip of the nanocapillary drifts by applying voltage, so that the control on the liquid at the tip of the nanocapillary is realized. By using the electrochemical injection method, the ultramicro-volume matrix molecules and the nanometer material solution are injected into the cells, so that secondary ion signals of amiodarone and biomolecules in the MCF-7 cells are enhanced.

Description

Technical Field

[0001] The present invention relates to the field of biomedical engineering, and in particular to a method for enhancing the sensitivity of ToF-SIMS detection in single cells. Background Art

[0002] Single-cell analysis plays a crucial role in biological research. Because individual cells exhibit heterogeneity in structure, chemical composition, and metabolism, analyzing the composition and abundance of intracellular components at the single-cell level is essential to reveal their structure and function in biological systems. Currently, single-cell analysis methods include single-cell fluorescence imaging, single-cell electrochemical analysis, and single-cell mass spectrometry. Compared to single-cell fluorescence and electrochemical analysis, single-cell mass spectrometry offers the advantages of being label-free and capable of imaging multiple components. Single-cell mass spectrometry imaging techniques include matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-ToF-MS), desorption electrospray ionization mass spectrometry (DESI-MS), and time-of-flight secondary ion mass spectrometry (ToF-SIMS). However, MALDI-ToF-MS and DESI-MS are more suitable for tissue imaging due to their micron-level spatial resolution. SIMS exhibits the highest lateral resolution of all mass spectrometry imaging methods. ToF-SIMS, with its high submicron spatial resolution, is well-suited for single-cell and subcellular imaging. ToF-SIMS has become an important tool for label-free, high-resolution chemical imaging to visualize the spatial distribution of substances associated with single cells. With its widespread application in pharmacology, the technique covers a wide range of studies, from determining the spatial colocalization of drugs and biomolecules to studying the diffusion and distribution of drugs in single cells and tissues.

[0003] However, the low secondary ion yield of biomolecules has been a major limitation to their application. To improve secondary ion yield, research has focused on novel ion sources, laser-assisted secondary ionization, and matrix-enhanced effects. For matrix-enhanced effects, noble metals, graphene oxide, and matrix molecules have been applied to single cell surfaces. However, the matrix-enhanced effect is limited in its inability to increase secondary ion yield for components with complex intracellular chemical environments and severe matrix effects.

[0004] Recent studies have shown that intracellular metabolites are also used to depict cell phenotypes. If these nanomaterials or molecules are taken up by cells as matrices, they may enhance the signals of intracellular components, thereby obtaining more in-depth metabolic and phenotypic information in single cells. Some important matrices cannot cross the cell membrane, or the use of these matrices during cell culture will change the physiological state of the cell. How to ensure that these matrices can quickly enter the cytoplasm is a key issue. In cell biology, when certain matrices need to be introduced into the cytoplasm of living cells, patch clamp technology is usually used. The commonly used microinjection method is puncture injection, that is, by quickly inserting a microcapillary into the cell, injecting a certain amount of material, and then withdrawing it. However, due to the large tip of the microcapillary and the injected matrix, this method has some important problems: (1) the large tip can damage the cell even when the cell is dead; (2) the common patch clamp method is difficult to control the high volume of the injected solution and difficult to successfully inject; (3) when the capillary tip is inserted into the cell, it is difficult to control its position, which reduces the success rate of injection. Nanocapillaries can reduce damage to cells and control the position of the tip when inserted into cells, but injecting matrix into cells using nanocapillary patch clamps remains difficult. Summary of the Invention

[0005] In order to address the deficiencies of the prior art, the present invention discloses a method for enhancing the sensitivity of ToF-SIMS detection in single cells, which does not damage cells, is simple to inject with a high injection success rate, and is easy to control the position when the capillary tip is inserted into the cell.

[0006] The present invention discloses a method for enhancing the sensitivity of ToF-SIMS detection in a single cell, which comprises: S1: material preparation, preparing ITO glass and performing surface treatment on the ITO glass, preparing nanocapillaries and characterizing the nanocapillaries;

[0007] S2: Cell culture and inoculation: MCF-7 cells were inoculated on the ITO glass surface treated in S1 and cultured in culture medium;

[0008] S3: Cell treatment: The ITO glass inoculated with MCF-7 cells in S2 was treated with amiodarone, and the culture medium was replaced after treatment.

[0009] S4: Fill the nanocapillary with an organic solution, and absorb the quantum dots into the nanocapillary tip under the action of voltage. Then, insert the nanocapillary tip into a single cell on an ITO glass cover slip, and inject the quantum dot solution into the MCF-7 cells under the action of voltage.

[0010] S5: taking the ITO glass out of the culture medium, rinsing it, cleaning the edge of the ITO glass, and then freezing the ITO glass;

[0011] S6: Analysis of MCF-7 cells on ITO glass using ToF-SIMS.

[0012] Furthermore, the surface treatment of the ITO glass in S1 includes: first, cleaning the ITO glass with acetone, then washing the ITO glass with ethanol and pure water in sequence, performing ultrasonic cleaning for at least 30 minutes after each cleaning, then cleaning the ITO glass with a sulfuric acid-hydrogen peroxide mixed solution, and finally rinsing the ITO glass with pure water and drying it with nitrogen, and then sterilizing the dried ITO glass with an ultraviolet lamp for at least 30 minutes;

[0013] The sulfuric acid-hydrogen peroxide mixed solution contains sulfuric acid and 25-35% hydrogen peroxide in a weight ratio of 2.7-3.3:1.

[0014] Furthermore, the preparation and characterization of the nanocapillaries in S1 include: first cleaning the glass capillary with acetone, then washing it with ethanol and pure water in sequence, ultrasonically cleaning it for at least 30 minutes during the cleaning process, then blowing the glass capillary dry with nitrogen, and then using a laser drawer to make the dried glass capillary into a nanocapillary, and then the nanocapillary is silver-coated and characterized by field emission scanning electron microscopy.

[0015] Furthermore, the glass capillary has an outer diameter of 0.8-1.2 mm, an inner diameter of 0.6-0.8 mm, and a length of 7-8 cm.

[0016] Furthermore, the culture medium in S2 was RPMI-1640 medium supplemented with 7-13% fetal bovine serum and 80-120 μg mL -1 Streptomycin and 80-120 μg mL -1 The culture conditions are 35-39°C in a humidified environment containing 3-7% CO2 for 6-10 hours.

[0017] Furthermore, the concentration of amiodarone in S3 is 8-12 μg / mL, and the treatment time is 10-14 hours.

[0018] Furthermore, the quantum dots in S4 are amino-functionalized graphene quantum dots;

[0019] The organic solution in S4 is 1,2-dichloroethane solution. The nanocapillary absorbs quantum dots to the tip of the nanocapillary under the action of a voltage of -600--400mV, and the nanocapillary injects quantum dots into MCF-7 cells under the action of a voltage of +800-+1200mV.

[0020] Furthermore, in S5, the ITO glass is removed from the culture medium and rinsed in a 130-170 mM ammonium acetate solution at pH 7-7.8 for 25-35 seconds;

[0021] In S5, the ITO glass cover slip was quickly frozen in liquid propane at -196°C to -190°C, and the sample was freeze-dried for 4-8 hours.

[0022] Furthermore, all other reagents were of analytical grade, and all aqueous solutions were prepared using ultrapure water.

[0023] Beneficial effects of the present invention:

[0024] The present invention utilizes a nanocapillary filled with an organic solvent immiscible with a matrix solution. By applying a voltage between a working electrode inserted into the capillary and a counter electrode immersed in an external solution, the matrix solution at the nanocapillary tip drifts due to the potential difference between the two liquid phases, thereby achieving control of the liquid at the nanocapillary tip. This method involves applying a voltage at the liquid / liquid interface to alter surface tension; this driving force can induce the aqueous solution at the nanocapillary tip to enter and exit the capillary. When the nanocapillary tip, filled with an organic electrolyte solution, is immersed in the matrix solution using a micromanipulator, the matrix solution is drawn into the nanocapillary tip by applying a negative potential to the organic solution; it is expelled from the nanocapillary tip by applying a positive potential to the organic solution. Using the electrochemical injection method of the present invention, ultramicrovolumes of matrix molecules and nanomaterial solutions were injected into cells, resulting in enhanced secondary ion signals for amiodarone and biomolecules in MCF-7 cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Figure 2 is a ToF-SIMS positive ion mass spectrometry (a) and chemical imaging (b) of MCF-7 cells treated with amiodarone in an embodiment of the present application, including the deep profiling results of MCF-7 cells treated with amiodarone and the deep profiling results of MCF-7 cells treated with amiodarone after incubation with amino-polymerized graphene quantum dots.

[0026] Figure 2 This is the current trajectory curve of graphene quantum dots based on capillary in situ cell micro-nanoinjection in the embodiment of the present application.

[0027] Figure 3 These are the intracellular amiodarone mass spectra of cells without micro-nano injection of graphene quantum dots (A) and cells with micro-nano injection of graphene quantum dots (B) in the embodiments of the present application.

[0028] Figure 4These are mass spectrometry images of ribosome fragment ions, phospholipid head group ions and amiodarone quasi-molecular ions in cells without micro-nano injection of graphene quantum dots and cells with micro-nano injection of graphene quantum dots in an embodiment of the present application. DETAILED DESCRIPTION

[0029] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the specific implementation manner of the present invention will be clearly and completely described below.

[0030] The present invention discloses a method for enhancing the sensitivity of ToF-SIMS detection in single cells, comprising:

[0031] S1: Material preparation, preparation of ITO glass and surface treatment of ITO glass, preparation of nanocapillaries and characterization of nanocapillaries;

[0032] S2: Cell culture and inoculation: MCF-7 cells were inoculated on the ITO glass surface treated in S1 and cultured in culture medium;

[0033] S3: Cell treatment: The ITO glass inoculated with MCF-7 cells in S2 was treated with amiodarone, and the culture medium was replaced after treatment.

[0034] S4: Fill the nanocapillary with an organic solution, and absorb the quantum dots into the nanocapillary tip under the action of voltage. Then, insert the nanocapillary tip into a single cell on an ITO glass cover slip, and inject the quantum dot solution into the MCF-7 cells under the action of voltage.

[0035] S5: taking the ITO glass out of the culture medium, rinsing it, cleaning the edge of the ITO glass, and then freezing the ITO glass;

[0036] S6: Analysis of MCF-7 cells on ITO glass using ToF-SIMS.

[0037] The present invention ensures that the ITO glass surface is clean and hydrophilic by cleaning and surface activation, which is conducive to the uniform attachment of MCF-7 cells, avoids impurities interfering with subsequent detection signals, and provides a stable substrate for single-cell analysis. The preparation and characterization of nanocapillaries ensure that the tip size and physical properties of the nanocapillary are controllable, laying the foundation for subsequent precise injection into single cells, reducing cell damage and improving the success rate of the operation. The voltage gradient is used to control the absorption and injection of quantum dots by the nanocapillary to achieve quantitative and directional delivery in single cells, avoid mechanical damage or human operation errors in traditional microinjection, ensure that quantum dots are efficiently enriched in target cells, and enhance the detection signal intensity. The tiny pore size of the nanocapillary is combined with single-cell level operation to accurately locate the cytoplasm, achieve targeted labeling of subcellular structures, and improve signal specificity. By bypassing the averaging effect of multi-cell mixed detection, quantum dots are directly injected into single cells to avoid the dilution of signals due to cell heterogeneity, so that ToF-SIMS can capture the characteristic ion signals of trace substances such as amiodarone and its metabolites in single cells, thereby improving the detection sensitivity at the single-cell level. As a strong signal probe, quantum dots can be specifically bound to target molecules due to their surface modification or fluorescent properties. They can be precisely introduced into cells through nanocapillaries to amplify the detection signal and reduce background noise. Flushing removes culture medium residues and non-specifically adsorbed impurities to reduce background interference during mass spectrometry analysis; freezing treatment fixes cell morphology and molecular distribution to avoid degradation or diffusion of target substances during sample preparation, ensuring the integrity and authenticity of the ion signal. From material processing to cell culture, voltage-driven injection, and sample freezing, the present invention has clear parameters for each step, which facilitates laboratory reproduction, reduces the impact of human operational differences on the results, and ensures the stability of detection sensitivity. The present invention is suitable for adherent cells such as MCF-7 cells, and nanocapillary technology can be extended to other single-cell models. Combined with the high resolution of ToF-SIMS, it provides a universal platform for the analysis of trace substances such as small molecules and drug metabolites in single cells.

[0038] As an embodiment, the surface treatment of the ITO glass in S1 includes: first, cleaning the ITO glass with acetone, then washing the ITO glass with ethanol and pure water in sequence, performing ultrasonic cleaning for at least 30 minutes after each cleaning, then cleaning the ITO glass with a sulfuric acid-hydrogen peroxide mixed solution, and finally rinsing the ITO glass with pure water and drying it with nitrogen, and then sterilizing the dried ITO glass with an ultraviolet lamp for at least 30 minutes;

[0039] The sulfuric acid-hydrogen peroxide mixture consists of sulfuric acid and 25-35% hydrogen peroxide in a ratio of 2.7-3.3 parts by weight:1. Acetone, as a strong organic solvent, quickly dissolves and removes grease, wax, and organic contaminants from the ITO glass surface, paving the way for subsequent polar solvent cleaning and preventing the formation of a protective layer of organic impurities that could hinder subsequent steps. Ethanol, a moderately polar solvent, dissolves residual acetone and some polar organic matter while also reducing surface tension, allowing pure water to more easily penetrate tiny pores. The combination of sulfuric acid and hydrogen peroxide produces strong oxidizing species that completely oxidize residual organic matter into CO2 and H2O. Excessive sulfuric acid ensures a strong acidic solution, maintaining the oxidation reaction and preventing excessive H2O2 from rapidly decomposing the solution and generating large bubbles. This ensures a gentle, controllable cleaning process that does not damage the ITO layer. The oxidation reaction introduces a large number of hydroxyl and carboxyl groups to the glass surface, transforming the surface from hydrophobic to strongly hydrophilic. The hydrophilic surface promotes the adsorption of adhesion proteins of MCF-7 cells, ensures uniform cell spreading, reduces cell aggregation or shedding caused by surface hydrophobicity, and provides a stable substrate for subsequent single-cell manipulation. + or SO4 2- Ions affect the pH of the culture medium during cell culture. Nitrogen drying prevents water marks caused by natural drying, while airflow purges further remove microparticles. Ultraviolet disinfection prevents abnormal cell status caused by bacterial contamination, ensuring the authenticity and reliability of the amiodarone-treated cell response in S3. The sterile environment reduces interference of microbial metabolites with the mass spectrometry signal during subsequent ToF-SIMS analysis, thereby reducing background noise.

[0040] As an embodiment, the preparation and characterization of the nanocapillaries in S1 include: first, cleaning the glass capillary with acetone, then washing it with ethanol and pure water, followed by ultrasonic cleaning for at least 30 minutes during the cleaning process. The glass capillary is then blown dry with nitrogen. The dried glass capillary is then formed into a nanocapillary using a laser drawer. The nanocapillary is then silver-coated and characterized using a field emission scanning electron microscope. Acetone dissolves organic contaminants such as grease, release agent, and fingerprint residue on the surface of the glass capillary. Residual impurities such as these can affect the uniformity of the subsequent silver coating. Ethanol, as a polar solvent, removes residual acetone and reduces surface tension, allowing pure water to more easily penetrate the tiny pores on the inner wall of the capillary. Ultrasonic cleaning removes inorganic particles, especially nanoscale contaminants, from the glass surface and the tube through the cavitation effect, preventing clogging of the capillary tip or contamination of the quantum dot solution. Nitrogen drying thoroughly removes moisture from the inside and outside of the capillary to prevent residual water from vaporizing due to high temperature during laser drawing and forming bubbles, which can lead to uneven tip structure. Drying the surface ensures a tight bond between the subsequent silver coating and the glass substrate. Laser heating of glass capillaries and application of tension can produce uniform thin tubes with a tip diameter of 10-100nm, which meets the minimally invasive requirements of single-cell operations. A tip that is too thick can cause damage to the cell membrane or cell rupture, while a tip that is too thin may make it difficult to push out the solution due to high resistance. The laser-drawn capillary tip has a smooth, symmetrical conical shape, which reduces the mechanical resistance when inserted into the cell, reduces the physical damage to the phospholipid bilayer of the cell membrane, and improves the success rate of single-cell injection. Silver is a highly conductive material. After coating, the tip of the nanocapillary becomes a conductive electrode, which realizes electrically driven solution manipulation in the S4 voltage operation, stably absorbs quantum dot solutions through electroosmosis or electrophoresis at a voltage of -500mV, and accurately pushes out the solution through an electrically driven force at +1000mV. The silver coating isolates the glass from direct contact with the solution, avoiding the ionization of silicon hydroxyl groups on the glass surface under high voltage to produce OH - ions, maintaining the solution pH stability and the fluorescence performance of quantum dots. High-resolution FE-SEM imaging is used to confirm whether the nanocapillary tip diameter is within the target range, ensuring that the injection can penetrate the cell membrane without causing irreversible damage.

[0041] As an embodiment, the outer diameter of the glass capillary is 0.8-1.2mm, the inner diameter is 0.6-0.8mm, and the length is 7-8cm. The wall thickness difference between the outer diameter and the inner diameter is 0.2-0.4mm, which ensures that the outer layer of the glass capillary maintains structural support during laser heating and drawing, avoiding overall softening and collapse during heating due to the wall being too thin, and the inner layer is easily melted and stretched to form a uniform nanotip. The inner diameter of 0.6-0.8mm determines the initial taper starting point of the nanocapillary after drawing. If the inner diameter is too small, the tip is prone to break due to stress concentration during drawing. If it is too large, the lower limit of the tip diameter is difficult to reach the 50-100nm required for single-cell operation. The outer diameter of 0.8-1.2mm is suitable for stiffness. Capillaries less than 0.8mm are prone to bending, especially after filling with solution, resulting in tip deviation during drawing or inaccurate positioning when inserted into cells. If it is greater than 1.2mm, the stiffness is too high, and laser heating requires higher power. Moreover, when inserted into cells, it is easy to puncture the cell membrane due to excessive rigidity. If the inner diameter is too thin, the resistance to solution flow will increase, resulting in delayed solution delivery or excessive pressure during voltage drive. If the inner diameter is too thick, the nanocapillary will have too much liquid storage capacity.

[0042] As an embodiment, the culture medium in S2 is RPMI-1640 culture medium with 7-13% fetal bovine serum and 80-120 μg·mL -1 Streptomycin and 80-120 μg mL -1Penicillin is added, and the culture conditions are culturing at 35-39°C in a humidified environment containing 3-7% CO2 for 6-10 hours. RPMI-1640 medium is a commonly used basal culture medium for mammalian cells. It is rich in amino acids, vitamins, inorganic salts, etc., which can provide cells with a stable nutritional environment and support cell adherence growth and metabolic activity. Serum contains growth factors, hormones, adhesion factors and nutrients necessary for cell growth, which can promote the adhesion, proliferation and survival of MCF-7 cells. The concentration range is advantageous. 7-13% is a regular serum concentration, which can avoid nutritional deficiencies caused by low concentrations and prevent excessive cell proliferation or background interference caused by high serum concentrations. Streptomycin and penicillin are both broad-spectrum antibiotics that inhibit bacterial protein synthesis and cell wall synthesis, respectively. They can effectively prevent bacterial contamination, maintain the sterility of the cell culture environment, and avoid cell death or experimental errors caused by contamination. This concentration range can inhibit common bacteria without being toxic to cells due to excessive concentrations. CO2 is used to maintain the pH of the culture medium. Working in conjunction with the bicarbonate buffer system, it prevents the culture medium from becoming overly acidic or overly alkaline, ensuring stable enzyme activity and metabolism within the cells. The optimal temperature for human cells is 37°C. This range, centered around 37°C, allows for some fluctuation to avoid excessively high or low temperatures and ensure cell viability and proliferation. MCF-7 cells are adherent cells, and 6-10 hours ensures complete cell attachment without entering the logarithmic growth phase. This maintains a uniform cell state, facilitating subsequent amiodarone treatment or quantum dot injection. Too short a time may result in incomplete attachment, while too long may lead to excessive cell density and accumulation of metabolic waste, compromising cell health and experimental reproducibility.

[0043] As one embodiment, the amiodarone concentration in S3 is 8-12 μg / mL, and the treatment time is 10-14 hours. Amiodarone uptake by MCF-7 cells is a process mediated by passive diffusion or specific transporters. A concentration of 8-12 μg / mL is within the linear kinetic range of its transmembrane transport, resulting in a stable drug uptake rate. The intracellular drug concentration is positively correlated with the culture medium concentration, facilitating experimental reproducibility. A treatment period of 0-14 hours is sufficient for amiodarone to cross the cell membrane and reach equilibrium within the cell, thus avoiding insufficient drug uptake caused by short treatment times. The MCF-7 cell cycle lasts approximately 20-24 hours. A 10-14 hour treatment period places the cell in interphase, when cells are metabolically active but not yet dividing. This allows for more uniform drug action and reduces fluctuations in the detection signal caused by differences in cell cycle stages. Exceeding 14 hours may result in excessive intracellular drug accumulation, triggering oxidative stress or organelle damage, and compromising the success rate of subsequent quantum dot injection procedures. This timeframe ensures that cells retain intact membrane structure and physiological activity after treatment, facilitating precise manipulation within single cells.

[0044] As an embodiment, the quantum dots in S4 are amino-functionalized graphene quantum dots. The organic solution in S4 is a 1,2-dichloroethane solution. The nanocapillary absorbs the quantum dots into the tip of the nanocapillary under the action of a voltage of -600--400mV, and the nanocapillary injects the quantum dots into MCF-7 cells under the action of a voltage of +800-+1200mV. The amino groups on the surface of the amino-functionalized graphene quantum dots can reduce the cytotoxicity of the quantum dots, making them more biocompatible with MCF-7 cells. In the process of injecting quantum dots into cells, the damage to the cells is small, which can ensure that the cells can still maintain normal physiological functions and activities after the injection of quantum dots, which is conducive to the subsequent accurate ToF-SIMS detection of cells and avoids abnormal test results due to cell damage. Amino group is an active functional group that can be easily covalently linked to other biological molecules. In experiments, this property allows quantum dots to be used as a multifunctional carrier, not only for intracellular imaging, but also to combine with specific biological molecules to achieve targeted labeling of specific molecules or structures within cells, thereby improving the specificity and accuracy of ToF-SIMS detection. Graphene quantum dots have excellent optical and electrical properties, such as fluorescence emission characteristics. In ToF-SIMS detection, these properties can serve as an additional signal source, complementing traditional secondary ion signals, to provide more information for analyzing the molecular composition and distribution within cells, and help to more fully understand the physiological state of cells and the mechanism of drug action. 1,2-Dichloroethane is an organic solvent with good insulating properties. Filling the nanocapillary with this solution can effectively isolate the quantum dot solution from the external electric field, avoid short circuits during the absorption and injection of quantum dots, and ensure that the voltage can stably act on the quantum dot solution, thereby achieving precise control of the quantum dot absorption and injection process. The viscosity and fluidity of 1,2-dichloroethane are moderate, ensuring smooth flow of the quantum dot solution within the nanocapillary while preventing uncontrolled outflow of the solution due to excessive fluidity. This allows the quantum dot solution to be drawn into the nanocapillary tip as intended under the influence of voltage and accurately injected into MCF-7 cells. A voltage of -600--400mV is used to draw the quantum dots. Within this negative voltage range, the nanocapillary tip generates an electric field that attracts the positively charged amino-functionalized graphene quantum dots toward the nanocapillary tip, thereby drawing the quantum dots into the nanocapillary tip. This electrically driven drawing method offers high controllability and accuracy, allowing precise control of the amount of quantum dots drawn, preventing the subsequent experimental results from being affected by drawing in too many or too few quantum dots. A voltage of +800-+1200mV is used to inject quantum dots. Under the action of positive voltage, the tip of the nanocapillary will generate an electric field force in the opposite direction, pushing out the absorbed quantum dot solution and injecting it into MCF-7 cells.This higher positive voltage range can provide sufficient driving force to enable the quantum dot solution to overcome the resistance of the cell membrane and smoothly enter the interior of the cell. At the same time, by precisely controlling the magnitude and application time of the voltage, the amount of injected quantum dots and the injection speed can be further controlled to ensure that the damage to the cells during the injection process is minimized. In summary, the use of amino-functionalized graphene quantum dots, 1,2-dichloroethane solution and specific voltage parameters can improve the accuracy and controllability of quantum dot absorption and injection, ensure the activity and integrity of the cells, and provide strong support for enhancing the sensitivity of ToF-SIMS detection in single cells.

[0045] As an embodiment, in S5, the ITO glass is removed from the culture medium and rinsed in a 130-170mM ammonium acetate solution with a pH of 7-7.8 for 25-35 seconds. In S5, the ITO glass cover slip is quickly frozen in liquid propane at -196°C--190°C, and the sample is freeze-dried for 4-8 hours. The neutral pH environment of 7-7.8 is close to the physiological pH of the human body, which avoids the damage of strong acid / strong base to cell surface molecules and maintains cell integrity and molecular activity. The physiological isotonic concentration of 130-170mM is close to the osmotic pressure of the extracellular fluid, which prevents the expansion or contraction of cells due to changes in osmotic pressure, avoids cell membrane rupture or cell morphology changes, and ensures the authenticity of the cell structure in subsequent tests. As a polar solvent, ammonium acetate can effectively remove residual components such as fetal bovine serum and antibiotics in the culture medium, and avoid these organic substances from generating interfering signals in subsequent tests. NH4 formed after the dissociation of ammonium acetate + and CH3COO - Ions neutralize static charges on the ITO glass and cell surfaces, reducing electrostatic adsorption on the sample surface, preventing secondary ion scattering and signal distortion during detection, and improving ToF-SIMS imaging resolution and data accuracy. A 25-35 second rinse time fully removes impurities without compromising cell adhesion to the ITO glass substrate due to prolonged rinsing, ensuring that single-cell samples remain fixed in place during subsequent processing. Ultra-low temperatures, approaching liquid nitrogen's -196°C, instantly transform intracellular water into amorphous vitreous ice, preventing mechanical damage to cell membranes and organelles caused by ice crystal growth during traditional slow freezing processes and maximizing the preservation of the in-situ distribution of intracellular molecules. Liquid propane, with a boiling point of approximately -42°C, is used as the freezing medium. Pre-cooling in liquid nitrogen allows for rapid heat transfer, resulting in a uniform and extremely fast freezing rate, ensuring complete vitrification of water within single cells and preventing damage to cell structure caused by ice crystals. Low-temperature drying for 4-8 hours, under vacuum, allows for direct sublimation of the vitreous ice to remove water, avoiding molecular degradation or cell structural collapse associated with normal-temperature drying. The surface of the dried sample is smoother, which reduces the scattering of the ion beam by water, improves the yield of secondary ions and detection sensitivity, avoids sample contamination or discharge caused by residual moisture, and ensures the stability and repeatability of the mass spectrometry signal.

[0046] For comparison, the MCF-7 cells treated with amiodarone were incubated in 80 μg / mL amino-GEQDs culture medium. In order to better compare the enhancing effect of the injection of amino-GEQDs, the MCF-7 cells at this time were inoculated and cultured on a non-conductive glass substrate. Before ToF-SIMS depth analysis, the cell samples were quickly frozen and fixed with liquid nitrogen, and then vacuum freeze-dried. ToF-SIMS depth analysis positive ion mass spectrometry and chemical imaging of MCF-7 cells treated with amiodarone without incubation of amino-GEQDs and incubated with amino-GEQDs are shown in Figure 2. Figure 1 shown.

[0047] Using the electrochemical injection method, a nanocapillary pulled from a quartz capillary was used as a tool to precisely inject graphene quantum dots, which served as a matrix, into specific locations of single cells, thereby enhancing the secondary ion signal of amiodarone within the cells. In actual experiments, the ToF-SIMS signal of the mixture of graphene quantum dots and amiodarone was observed on a glass slide to be amplified by 160 times, which is significantly higher than other signal enhancement materials previously reported. Graphene quantum dots can enhance the signal of cellular components in the high mass-to-charge ratio range. Graphene quantum dots can serve as a good matrix molecule to enhance the signal of amiodarone within cells. The diameter of the nanocapillary tip is about 100 nanometers, which significantly reduces the damage to single cells when the nanocapillary is inserted into the cell.

[0048] Before inserting the nanocapillary into a cell, it must be secured to the holder of a micromanipulator. The nanocapillary, filled with 1,2-dichloroethane solution, is mounted on a micromanipulator arm whose position can be adjusted and controlled. Images of the nanocapillary are acquired using brightfield microscopy. The nanocapillary is secured to the microscope objective stage via the holder of the micromanipulator arm, and its movement is controlled by a motorized drive system. When the nanocapillary tip is immersed in a matrix aqueous solution via the micromanipulator, the matrix aqueous solution is drawn into the nanocapillary tip under the influence of a negative potential applied in the organic solution. During DC potential control of liquid injection or ejection, the nanocapillary tip, connected to an Ag / AgCl electrode, serves as the working electrode and is inserted into the graphene quantum dot solution at the nanocapillary tip filled with 1,2-dichloroethane solution. Another Ag / AgCl electrode, immersed in the graphene quantum dot solution, serves as the counter electrode. After the nanocapillary tip was immersed in the graphene quantum dot solution, the interface voltage was maintained at -500 mV to encourage the graphene quantum dot solution to enter the nanocapillary. Once the nanocapillary was sufficiently filled with the graphene quantum dot solution, the nanocapillary tip, filled with the graphene quantum dot solution and 1,2-dichloroethane solution, was inserted into a single live MCF-7 cell. Brightfield microscopy also revealed the interface between the graphene quantum dot solution and the 1,2-dichloroethane solution in the nanocapillary. Subsequently, single cells of interest were gradually selected using white light transmission microscopy on an inverted microscope stage, and the motorized micromanipulator was adjusted and controlled. The nanocapillary tip was then removed from the graphene quantum dot solution and inserted into the cell culture medium. To ensure that the graphene quantum dot solution filling the nanocapillary tip was not lost during this movement, the voltage was reduced to -100 mV. This flow was stopped by gradually adjusting the organic phase potential to approximately -100 mV.

[0049] As long as the potential is maintained at -100mV, the volume of the aqueous solution in the capillary remains constant. Subsequently, a potential is maintained that can support the volume of the graphene quantum dot solution in the tip of the nanocapillary. The cell culture dish is placed on a bright field microscope platform, and a single cell is selected on an ITO coverslip. This injection and ejection process cannot be repeated with the same nanocapillary, so a nanocapillary with a tip filled with graphene quantum dot solution is inserted into living MCF-7 cells. The tip of the nanocapillary is about 100 nanometers, much smaller than the standard microcapillary used for patch clamping, which is particularly conducive to insertion into cells. Figure 2 As shown in Figure 2, the nanocapillary was exposed to a cell culture medium containing approximately 150 mM inorganic cations or anions, generating a current of approximately -600 pA. After the nanocapillary tip was injected into the cell membrane using a micromanipulator, the current fluctuated and decayed. Figure 2As shown in the blue inset, the current decreases for a period of time because the conductivity of the cell membrane is much lower than that of the cell culture solution. When the nanocapillary tip is inserted into the cell, the cell membrane is located between the nanocapillary tip and the counter electrode. Subsequently, the potential is adjusted back to +1000mV, the aqueous solution is completely removed from the nanocapillary and quickly injected into the cell. During this period, the current immediately increases to +10000pA. Then, as the graphene quantum dot solution is injected, the current still fluctuates and decays, as shown in Figure 2. Figure 2 As shown. Under the action of the electric field, the graphene quantum dot solution can be injected into the cell by changing the surface tension of the liquid / liquid interface, and this process can be observed under a bright field microscope. During this process, as the solution is injected, the volume of the injected cell gradually increases, as shown in Figure 2. Figure 3 At the same time, only cells injected with graphene quantum dot solution will glow under fluorescence, as shown in Figure 4 However, excessive amounts of graphene quantum dot solution must not be injected into the cells to ensure cell viability after injection. When the nanocapillary tip is removed from the cell, the current increases briefly because the conical shape of the nanocapillary tip increases contact with the cell culture medium as it begins to leave the cell. The current increases to its maximum value until the nanocapillary tip is removed from the cell membrane. The maximum current remains constant while the nanocapillary tip is removed from the cell culture medium, but the current instantly decays to 0 pA at the moment the nanocapillary tip is removed from the cell culture medium.

[0050] It should be understood that those skilled in the art can make improvements or changes based on the above description, and all such improvements and changes should fall within the scope of protection of the appended claims of the present invention.

Claims

1. A method for enhancing the sensitivity of ToF-SIMS detection in single cells, characterized in that: include: S1: Material preparation, preparation of ITO glass and surface treatment of ITO glass, preparation of nanocapillaries and characterization of nanocapillaries; S2: Cell culture and inoculation: MCF-7 cells were inoculated on the ITO glass surface treated in S1 and cultured in culture medium; S3: Cell treatment: The ITO glass inoculated with MCF-7 cells in S2 was treated with amiodarone, and the culture medium was replaced after treatment. S4: Fill the nanocapillary with an organic solution, and absorb the quantum dots into the nanocapillary tip under the action of voltage. Then, insert the nanocapillary tip into a single cell on an ITO glass cover slip, and inject the quantum dot solution into the MCF-7 cells under the action of voltage. S5: taking the ITO glass out of the culture medium, rinsing it, cleaning the edge of the ITO glass, and then freezing the ITO glass; S6: Analysis of MCF-7 cells on ITO glass using ToF-SIMS.

2. The method for enhancing the sensitivity of ToF-SIMS detection in single cells according to claim 1, characterized in that: The surface treatment of the ITO glass in S1 includes: first, cleaning the ITO glass with acetone, then washing the ITO glass with ethanol and pure water in sequence, performing ultrasonic cleaning for at least 30 minutes after each cleaning, then cleaning the ITO glass with a sulfuric acid-hydrogen peroxide mixed solution, and finally rinsing the ITO glass with pure water and drying it with nitrogen. After drying, the ITO glass is sterilized with an ultraviolet lamp for at least 30 minutes. The sulfuric acid-hydrogen peroxide mixed solution contains sulfuric acid and 25-35% hydrogen peroxide in a weight ratio of 2.7-3.3:

1.

3. The method for enhancing the sensitivity of ToF-SIMS detection in single cells according to claim 1, characterized in that: The preparation and characterization of the nanocapillaries in S1 include: first, cleaning the glass capillaries with acetone, then washing with ethanol and pure water in sequence, ultrasonically cleaning for at least 30 minutes during the cleaning process, then blowing the glass capillaries dry with nitrogen, and then using a laser drawer to make the dried glass capillaries into nanocapillaries. The nanocapillaries are then silver-coated and characterized by field-emission scanning electron microscopy.

4. The method for enhancing the sensitivity of ToF-SIMS detection in single cells according to claim 3, characterized in that: The outer diameter of the glass capillary is 0.8-1.2 mm, the inner diameter is 0.6-0.8 mm, and the length is 7-8 cm.

5. The method for enhancing the sensitivity of ToF-SIMS detection in single cells according to claim 1, characterized in that: The culture medium in S2 is RPMI-1640 medium supplemented with 7-13% fetal bovine serum and 80-120 μg mL -1 Streptomycin and 80-120 μg mL -1 The culture conditions are 35-39°C in a humidified environment containing 3-7% CO2 for 6-10 hours.

6. The method for enhancing the sensitivity of ToF-SIMS detection in single cells according to claim 1, characterized in that: The concentration of amiodarone in S3 was 8-12 μg / mL, and the treatment time was 10-14 hours.

7. The method for enhancing the sensitivity of single-cell ToF-SIMS detection according to claim 1, characterized in that: The quantum dots in S4 are amino-functionalized graphene quantum dots; The organic solution in S4 is 1,2-dichloroethane solution. The nanocapillary absorbs quantum dots to the tip of the nanocapillary under the action of a voltage of -600--400mV, and the nanocapillary injects quantum dots into MCF-7 cells under the action of a voltage of +800-+1200mV.

8. The method for enhancing the sensitivity of ToF-SIMS detection in single cells according to claim 1, characterized in that: In S5, the ITO glass was removed from the culture medium and rinsed in a 130-170 mM ammonium acetate solution at pH 7-7.8 for 25-35 seconds; In S5, the ITO glass cover slip was quickly frozen in liquid propane at -196°C to -190°C, and the sample was freeze-dried for 4-8 hours.

9. The method for enhancing the sensitivity of single-cell ToF-SIMS detection according to claim 1, characterized in that: All other reagents were of analytical grade, and all aqueous solutions were prepared using ultrapure water.

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