A method for preparing a single cell smear suitable for matrix assisted laser desorption ionization

Abstract

The application discloses a preparation method of a single cell smear suitable for matrix assisted laser desorption ionization, and comprises the following steps: S1, centrifuging a cell suspension to remove supernatant, adding a formic acid ammonium solution containing bovine serum albumin, stirring uniformly, and obtaining a pretreated solution; S2, precooling an indium tin oxide glass slide, coating the pretreated solution on the glass slide, primary baking, cleaning with the formic acid ammonium solution, and secondary baking, and obtaining the single cell smear. The application has the advantages of improving the interface spreading effect of the cell suspension on the ITO glass slide and avoiding the inhibition and interference of residues on the detection signal of the matrix assisted laser desorption ionization mass spectrum.

Description

Technical Field

[0001] This application relates to the field of single-cell metabolomics technology, and in particular to a method for preparing single-cell smears suitable for matrix-assisted laser desorption / ionization. Background Technology

[0002] Matrix-assisted laser desorption / ionization (MALDI) technology, as a high-throughput, label-free, and highly sensitive analytical method, has been widely applied in proteomics, metabolomics, and spatial omics research. MALDI mass spectrometry enables in-situ detection at the tissue section or single-cell level. Combined with mass spectrometry imaging, it can simultaneously acquire spatial distribution information of multiple metabolites, offering significant advantages such as fast analysis speed, low sample consumption, and no need for complex pretreatment. In the field of single-cell metabolomics, MALDI mass spectrometry has become one of the core analytical tools due to its ability to desorb metabolic heterogeneity between cells, reveal changes in disease-related metabolic pathways, and discover novel biomarkers.

[0003] Sample preparation is a crucial prerequisite for MALDI single-cell metabolomics analysis, and its quality directly affects the sensitivity, spatial resolution, and metabolite fidelity of mass spectrometry detection. The commonly used MALDI detection substrate is indium tin oxide (ITO) slides, which have a hydrophobic surface and poor spreading properties for aqueous cell suspensions. An ideal single-cell smear should achieve a uniform, monolayer, and in-situ conformal distribution of cells on the slide surface, avoiding cell overlap, metabolite diffusion, and morphological damage, thereby ensuring the accuracy and reproducibility of subsequent mass spectrometry imaging.

[0004] Currently, conventional methods for preparing single-cell smears mainly involve resuspending tissue-derived single cells in volatile salt solutions (such as ammonium formate solution) and directly coating them onto the surface of ITO slides. While this method is simple to operate, it faces significant technical challenges: due to the low surface free energy of ITO slides, ammonium formate solution tends to shrink into hemispherical droplets on the slide, resulting in a large contact angle and preventing the cell suspension from spreading evenly. During solvent evaporation, capillary flow at the droplet edges triggers the "coffee ring effect," causing cells to aggregate and overlap at the edges. Simultaneously, intracellular metabolites easily diffuse with the liquid flow, leading to morphological damage and loss of metabolic information. These shortcomings severely limit the detection performance of MALDI mass spectrometry at the single-cell level.

[0005] To address these issues, existing technologies attempt to pretreat ITO slides with surface modifiers such as poly-L-lysine, sodium carboxymethyl cellulose, or dextran to improve cell adhesion and spreading. However, these conventional spreading or adhesive agents inevitably form dense envelopes around cells, severely hindering the subsequent co-crystallization process of MALDI matrix and intracellular metabolites, leading to significant ion inhibition and signal loss, thus affecting the sensitivity and accuracy of metabolite detection. Furthermore, repeated washing steps may cause significant cell detachment, reducing detection throughput. Summary of the Invention

[0006] To improve the interfacial spreading performance of cell suspensions on hydrophobic ITO slides, this application provides a method for preparing single-cell smears suitable for matrix-assisted laser desorption / ionization.

[0007] In a first aspect, this application provides a method for preparing single-cell smears suitable for matrix-assisted laser desorption / ionization, employing the following technical solution: A method for preparing single-cell smears suitable for matrix-assisted laser desorption / ionization includes the following steps: S1. Centrifuge the cell suspension to remove the supernatant, add ammonium formate solution containing bovine serum albumin, stir well to obtain pretreatment solution; S2. Pre-cool the ITO slide, then coat it with a pretreatment solution, bake it initially, wash it with ammonium formate solution, bake it again, and obtain a single-cell smear.

[0008] By employing the above technical solution, bovine serum albumin, as an amphiphilic protein, adsorbs at the gas-liquid and solid-liquid interfaces, reducing the surface tension of the pretreatment solution and the solid-liquid interfacial energy on the ITO slide surface, providing a thermodynamic driving force for the spreading of the cell suspension on the hydrophobic ITO slide surface. Simultaneously, the pre-cooling treatment of the ITO slide reduces the solvent evaporation rate on its surface, extending the kinetic time window for liquid film spreading, allowing the pretreatment solution to fully cover the target coating area before evaporation is complete. The initial baking-washing-rebaking steps enable rapid solvent evaporation, facilitating the rapid fixation of uniformly distributed cells and creating an ideal co-crystallization microenvironment for subsequent MALDI matrix spraying, thereby promoting the in-situ extraction and ionization efficiency of metabolites. The above steps work synergistically: bovine serum albumin provides the spreading driving force, pre-cooling provides the spreading time, and the initial baking-washing-rebaking steps rapidly fix uniformly distributed cells and construct a co-crystallization microenvironment, collectively achieving uniform spreading of the cell suspension on the ITO slide and the dispersion and sedimentation of single cells. Furthermore, pre-cooling inhibits the activity of residual enzymes within cells and reduces cell membrane permeability, thereby reducing enzymatic degradation and transmembrane leakage of metabolites during coating and helping to maintain the in-situ fidelity of intracellular metabolites. Initial drying is also performed. Bovine serum albumin, as a water-soluble protein, can be efficiently removed during subsequent washing with ammonium formate solution. Ammonium formate itself, as a volatile salt, further sublimates in the vacuum environment of mass spectrometry detection, thus achieving "end-to-end removability" of the smear surface. This avoids the inhibition and interference of residues on the matrix-assisted laser desorption / ionization mass spectrometry detection signal, ultimately resulting in a single-cell smear with uniform cell distribution, intact morphology, high metabolite fidelity, and a clean background.

[0009] Optionally, in step S1, the concentration of bovine serum albumin in the ammonium formate solution is 0.01-0.05% (w / v).

[0010] Optionally, in step S1, the concentration of bovine serum albumin in the ammonium formate solution is 0.02% (w / v).

[0011] By adopting the above technical solution, this application uses a specific concentration of bovine serum albumin and ammonium formate solution, which can ensure that bovine serum albumin can fully exert its interfacial regulation effect to achieve uniform spreading of cell suspension, and can also ensure that residual bovine serum albumin can be efficiently removed in a single subsequent wash. While maintaining a high cell retention rate and good cell morphology, the interference of smear background on matrix-assisted laser desorption / ionization mass spectrometry detection is minimized. When the concentration of bovine serum albumin (BSA) is too low, the amount of BSA adsorbed at the gas-liquid and solid-liquid interfaces is insufficient, which cannot effectively reduce the surface tension of the pretreatment solution. As a result, the cell suspension is still difficult to spread completely on the hydrophobic ITO slide surface, leading to uneven coverage and local cell aggregation. When the concentration of BSA is too high, the excess BSA cannot be completely removed in a single wash with ammonium formate solution, requiring more washes. The mechanical scouring force generated by multiple washes can cause the attached cells to detach, significantly reducing the cell survival rate. At the same time, the high concentration of BSA increases the colloidal osmotic pressure of the mixture, causing the cells to shrink and deform due to osmotic pressure imbalance, thus destroying the integrity of the cell morphology.

[0012] Optionally, the concentration of ammonium formate in the formate solution is 100-200 mmol / L.

[0013] By adopting the above technical solution, this application uses an ammonium formate solution of a specific concentration, which can provide isotonic conditions similar to the intracellular environment, maintain the normal morphology and membrane integrity of cells during coating and drying, effectively preserve the in-situ information of intracellular metabolites, and at the same time ensure that ammonium formate can be fully sublimated under the vacuum conditions of subsequent mass spectrometry detection without interfering with the mass spectrometry signal.

[0014] Optionally, in step S2, the pre-cooling temperature is 0-5℃.

[0015] Optionally, in step S2, the pre-cooling temperature is 4°C.

[0016] By adopting the above technical solution, this application uses a pre-cooling temperature of 4℃. On the one hand, this effectively reduces the solvent evaporation rate on the ITO slide surface while avoiding frost or condensation caused by excessively low temperatures. On the other hand, 4℃ is a commonly used low-temperature preservation temperature in the biological field. At this temperature, intracellular enzyme activity is sufficiently inhibited, and metabolic reactions are essentially halted, thus maximally preserving the original state and spatial distribution of intracellular metabolites and preventing enzymatic degradation or transmembrane diffusion of metabolites during coating. Simultaneously, the pretreatment solution at 4℃ has a suitable viscosity, preventing uneven cell distribution due to excessively low viscosity or excessively high viscosity that could hinder the spontaneous spreading of the liquid film. This facilitates uniform sedimentation and monolayer distribution of cells on the ITO slide surface.

[0017] Optionally, the initial baking temperature is 30-40℃, and the re-baking temperature is 30-40℃.

[0018] Optionally, the initial baking temperature is 37°C, and the re-baking temperature is 37°C.

[0019] By employing the above-mentioned technical solution, this application utilizes specific temperatures for initial baking and rebaking. This ensures a moderate evaporation rate, allowing the solvent to evaporate evenly within a short time, effectively inhibiting the diffusion and migration of metabolites and the coffee ring effect. Simultaneously, it maintains the integrity of cell morphology and membrane structure under mild conditions, maximizing the preservation of the original composition and spatial distribution information of intracellular metabolites. This provides a high-quality sample preparation foundation for subsequent matrix-assisted laser desorption / ionization mass spectrometry (MALADS) detection. During the initial baking process, trace amounts of bovine serum albumin form an extremely thin molecular buffer layer between the cells and the slide substrate. On one hand, this layer achieves in-situ 'micro-fixation' of single cells without damaging the cell membrane integrity, significantly improving cell survival rate. On the other hand, the clean buffer interface left after washing and desalting creates an ideal co-crystallization microenvironment for subsequent MALDI matrix spraying, greatly promoting the in-situ extraction and ionization efficiency of metabolites. The rebaking process after washing thoroughly removes background salts and leaves an ultrathin crystallization-promoting buffer layer composed of trace amounts of bovine serum albumin.

[0020] Optionally, the concentration of the cell suspension is 0.5 × 10⁻⁶. 6 -1.5×10 6 per mL.

[0021] By adopting the above technical solution, this application uses a cell suspension of a specific concentration, which enables cells to achieve ideal monolayer uniform dispersion on the surface of ITO glass slides, maintain sufficient spacing between adjacent cells to avoid signal crosstalk, and at the same time have a reasonable cell density to ensure sufficient data acquisition throughput, thereby facilitating subsequent high-quality single-cell metabolomics mass spectrometry imaging analysis.

[0022] Secondly, this application provides a single-cell smear suitable for matrix-assisted laser desorption / ionization, which is obtained by a method for preparing single-cell smears suitable for matrix-assisted laser desorption / ionization.

[0023] In summary, this application includes at least one of the following beneficial technical effects: 1. In this application, bovine serum albumin, as an amphiphilic protein, adsorbs at both the gas-liquid and solid-liquid interfaces, reducing the surface tension of the pretreatment solution and the solid-liquid interfacial energy on the ITO slide surface. This provides a thermodynamic driving force for the spreading of the cell suspension on the hydrophobic ITO slide surface. Simultaneously, the pre-cooling treatment of the ITO slide reduces the solvent evaporation rate on its surface, extending the kinetic time window for liquid film spreading, allowing the pretreatment solution to fully cover the target coating area before evaporation is complete. The initial baking-washing-rebaking steps enable rapid solvent evaporation, facilitating the rapid fixation of uniformly distributed cells and creating an ideal co-crystallization microenvironment for subsequent MALDI matrix spraying, thereby promoting the in-situ extraction and ionization efficiency of metabolites. The above steps work synergistically: bovine serum albumin provides the spreading driving force, pre-cooling provides the spreading time, and the initial baking-washing-rebaking steps rapidly fix uniformly distributed cells and construct a co-crystallization microenvironment, collectively achieving uniform spreading of the cell suspension on the ITO slide and dispersion and sedimentation of single cells. In addition, pre-cooling inhibits the activity of residual enzymes in cells and reduces cell membrane permeability, thereby reducing the enzymatic degradation and transmembrane leakage of metabolites during coating and helping to maintain the in-situ fidelity of intracellular metabolites; initial drying; and bovine serum albumin, as a water-soluble protein, can be efficiently removed during subsequent washing with ammonium formate solution. Ammonium formate itself, as a volatile salt, further sublimates in the vacuum environment of mass spectrometry detection, thus achieving "end-to-end removability" of the smear surface, avoiding the inhibition and interference of residues on matrix-assisted laser desorption / ionization mass spectrometry detection signals, and finally obtaining single-cell smears with uniform cell distribution, intact morphology, high metabolite fidelity and clean background; 2. This application uses a specific concentration of bovine serum albumin and ammonium formate solution, which can ensure that bovine serum albumin can fully exert its interfacial regulation effect to achieve uniform spreading of cell suspension, and can also ensure that residual bovine serum albumin can be efficiently removed in a single wash. While maintaining a high cell retention rate and good cell morphology, the interference of smear background on matrix-assisted laser desorption / ionization mass spectrometry detection is minimized. 3. This application employs a pre-cooling temperature of 4°C. On one hand, this effectively reduces the solvent evaporation rate on the ITO slide surface while preventing frost or condensation from forming on the slide surface due to excessively low temperatures. On the other hand, 4°C is a commonly used low-temperature storage temperature in the biological field. At this temperature, intracellular enzyme activity is sufficiently inhibited, and metabolic reactions are essentially halted, maximizing the preservation of the original state and spatial distribution of intracellular metabolites and preventing enzymatic degradation or transmembrane diffusion of metabolites during coating. Simultaneously, the pretreatment solution at 4°C has a suitable viscosity, preventing uneven cell distribution due to excessively low viscosity or hindering the spontaneous spreading of the liquid film due to excessively high viscosity. This facilitates uniform sedimentation and monolayer distribution of cells on the ITO slide surface. 4. This application employs specific temperatures for initial baking and rebaking, ensuring a moderate evaporation rate for uniform solvent drying within a short time, effectively inhibiting metabolite diffusion and migration and the coffee ring effect. Simultaneously, it maintains the integrity of cell morphology and membrane structure under mild conditions, maximizing the preservation of the original composition and spatial distribution information of intracellular metabolites, providing a high-quality sample preparation foundation for subsequent matrix-assisted laser desorption / ionization mass spectrometry (MALADS). During the initial baking process, trace amounts of bovine serum albumin form an extremely thin molecular buffer layer between the cells and the slide substrate. On one hand, this achieves in-situ 'micro-fixation' of single cells without damaging cell membrane integrity, significantly improving cell survival rate. On the other hand, the clean buffer interface left after washing and desalting creates an ideal co-crystallization microenvironment for subsequent MALDI matrix spraying, greatly promoting the in-situ extraction and ionization efficiency of metabolites. The rebaking process after washing thoroughly removes background salts, leaving an ultrathin crystallization-promoting buffer layer composed of trace amounts of bovine serum albumin. Attached Figure Description

[0024] Figure 1 This is a cell suspension spotting plate and ITO coated glass slide image from Example 1 of this application. Figure 1 A: Spot diagram of pure ammonium formate solvent system; Figure 1 B: Image of ITO glass slide coated with pure ammonium formate solvent system; Figure 1 C: TLC diagram of the ammonium formate solvent system containing bovine serum albumin; Figure 1 D: Image of ITO slide coated with a solution containing bovine serum albumin and ammonium formate; Figure 2 These are the cell smear effect image and bright-field high-resolution scan image from Example 1 of this application. Figure 2 A: Initial baking diagram of pure ammonium formate solvent system; Figure 2 B: Image of the re-baking process after cleaning with pure ammonium formate solvent system; Figure 2 C: Bright-field high-resolution scan image of the pure ammonium formate solvent system; Figure 2 D: Initial baking diagram of the ammonium formate solvent system containing bovine serum albumin; Figure 2 E: Image after cleaning and re-baking using a solvent system containing bovine serum albumin and ammonium formate; Figure 2 F: Bright-field high-resolution scan image of the ammonium formate solvent system containing bovine serum albumin; Figure 3 This is a bright-field high-resolution scan of the cell in Example 2 of this application. Figure 3 A: High-resolution bright-field scan of HeLa cells; Figure 3 B: High-resolution bright-field scan of mouse brain cells; Figure 3 C: High-resolution bright-field scan of mouse liver cells; Figure 4 This is a cell UMAP analysis diagram from Example 3 of this application. Figure 4A: HeLa cell UMAP analysis diagram; Figure 4 B: UMAP analysis diagram of mouse brain cells; Figure 5 These are bright-field high-resolution scans of cell smears with different treatments in Example 4 of this application; Figure 5 A: High-resolution bright-field scan of sodium carboxymethyl cellulose treated with sodium carboxymethyl cellulose; Figure 5 B: High-resolution bright-field scan of polylysine-treated image; Figure 5 C: High-resolution bright-field scan of dextran-treated image; Figure 5 D: High-resolution bright-field scan image treated with 0.02% bovine serum albumin; Figure 6 This is a cell signal intensity acquisition diagram of the single-cell smear protocol in Embodiment 4 of this application; Figure 7 These are bright-field high-resolution scans of cells with different concentrations of bovine serum albumin in Example 5 of this application; Figure 7 A: Image of an ITO-coated slide with a system without bovine serum albumin; Figure 7 B-7C: High-resolution bright-field scan of the system without added bovine serum albumin; Figure 7 D: Image of an ITO slide coated with an ammonium formate solution containing 0.01% (w / v) bovine serum albumin; Figure 7 E-7F: Bright-field high-resolution scan of an ammonium formate solution containing 0.01% (w / v) bovine serum albumin; Figure 7 G: Image of an ITO slide coated with an ammonium formate solution containing 0.02% (w / v) bovine serum albumin; Figure 7 H-7I: Bright-field high-resolution scan of an ammonium formate solution containing 0.02% (w / v) bovine serum albumin; Figure 7 J: Image of an ITO slide coated with an ammonium formate solution containing 0.05% (w / v) bovine serum albumin; Figure 7 K-7L: Bright-field high-resolution scan of an ammonium formate solution containing 0.05% (w / v) bovine serum albumin; Figure 7 M: An image of an ITO slide coated with an ammonium formate solution containing 0.1% (w / v) bovine serum albumin; Figure 7 N-7O: Bright-field high-resolution scanning image of an ammonium formate solution containing 0.1% (w / v) bovine serum albumin; Figure 8 These are bright-field high-resolution scans of cells with different coating treatments in Example 6 of this application; Figure 8 A: Bright-field high-resolution scan of cells in control group 1; Figure 8 B: High-resolution bright-field scans of cells from the control group 2. Detailed Implementation

[0025] The technical solutions in the embodiments of this application will be clearly and completely described below with reference to the embodiments of this application. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0026] This application discloses a method for preparing single-cell smears suitable for matrix-assisted laser desorption / ionization, comprising the following steps: S1, with a concentration of 0.5 × 10 6 -1.5×10 6 Centrifuge the cell suspension (cells / mL) to remove the supernatant, add ammonium formate solution containing bovine serum albumin, stir at 300-500 rpm for 3-5 min to obtain the pretreatment solution; S2. Pre-cool the ITO slide at 0-5℃, then coat it with a pretreatment solution, bake it at 30-40℃, clean it with ammonium formate solution, and bake it again at 30-40℃ to obtain a single-cell smear.

[0027] All raw materials used in the embodiments of this application are commercially available, wherein: Bovine serum albumin, Shanghai Aladdin Biochemical Technology Co., Ltd.; Ammonium formate, Shanghai Aladdin Biochemical Technology Co., Ltd.; Sodium carboxymethyl cellulose, Shanghai Aladdin Biochemical Technology Co., Ltd.; Poly-L-lysine, weight-average molecular weight 2000 Da, Shanghai Aladdin Biochemical Technology Co., Ltd. Dextran, weight average molecular weight 1000 Da, Shanghai Aladdin Biochemical Technology Co., Ltd.; HeLa cells, Shanghai Yubo Biotechnology Co., Ltd.; Mouse brain cells, C8-D1A, Shanghai Yaji Biotechnology Co., Ltd.; Mouse liver cells, Shenzhen Haodi Huatuo Biotechnology Co., Ltd. PBS solution, Shanghai Aladdin Biochemical Technology Co., Ltd.; ITO glass slides, Xi'an Qiyue Biotechnology Co., Ltd.

[0028] Example 1: Preparation of single-cell smears Bovine serum albumin (BSA) was added to a 150 mmol / L ammonium formate solution and stirred at 400 rpm for 4 min to prepare an ammonium formate solution containing 0.02% (w / v) BSA. HeLa cell suspension was then prepared using this 0.02% (w / v) BSA solution to a HeLa cell concentration of 1 × 10⁻⁶ cells / mL. 6The mixture was prepared at 1000 rpm for 4 min, and the supernatant was removed to obtain HeLa resuspended cells 1. The ITO slide was pre-cooled to 4°C, and 10 μL of HeLa resuspended cells 1 was dropped onto the ITO slide. (See figure...) Figure 1 C. Then, using a coating stick, the resuspended cells were evenly coated onto a pre-cooled ITO slide at 4°C, covering an area of ​​approximately 1.5 × 1.5 cm. (See attached image.) Figure 1 D. After observing the cell state, clumping rate, and uniformity of distribution on ITO slides coated with HeLa resuspended cells under a microscope, and confirming these parameters were correct, the slides were then baked at 37°C for 4 minutes. The results are shown in the figure. Figure 2 D. The dried smear area was cleaned with 150 mmol / L ammonium formate solution to remove residual salts, and the slide was then placed on a slide oven at 37°C for 4 minutes to obtain cell smear 1. The results are shown in [Figure 1]. Figure 2 E. Cell smear 1 was subjected to bright-field high-resolution scanning, and the results are shown in [the image]. Figure 2 F.

[0029] A separate control was prepared using ammonium formate solution without added bovine serum albumin for cell smears. The specific method was as follows: HeLa cell suspension was prepared to a concentration of 1×102 using 150 mmol / L ammonium formate solution. 6 The mixture was prepared at 1000 rpm for 4 min, and the supernatant was removed to obtain HeLa resuspended cells 2. The ITO slide was pre-cooled to 4°C, and 10 μL of HeLa resuspended cells 2 was dropped onto the ITO slide. (See figure...) Figure 1 A. Then, using a coating stick, the HeLa resuspended cells were evenly coated onto a pre-cooled ITO slide at 4°C, covering an area of ​​approximately 1.5 × 1.5 cm. (See...) Figure 1 B. After observing the cell state, clumping rate, and uniformity of distribution on ITO slides coated with HeLa resuspended cells under a microscope, and confirming these parameters were correct, the slides were then baked at 37°C for 4 minutes. The results are shown in the figure. Figure 2 A. The dried smear area was cleaned with 150 mmol / L ammonium formate solution to remove residual salts, and the slide was then placed on a slide oven at 37°C for 4 minutes to obtain cell smear 2. The results are shown in [Figure 2]. Figure 2 B. Cell smear 2 was subjected to bright-field high-resolution scanning, and the results are shown below. Figure 2 C.

[0030] Depend on Figure 1-2It is known that when using ammonium formate solution for cell smears, the hydrophobic surface of the ITO slide makes it difficult for the solution to spread evenly, easily causing local aggregation and uneven coating. Bright-field high-resolution scanning reveals that cells overlap and matrix crystals are present on the surface. However, when using ammonium formate solution containing bovine serum albumin for cell smears, the solution spreads evenly on the ITO slide, and bright-field high-resolution scanning shows that single cells are evenly distributed on the smear, with a clean surface and no obvious matrix crystals.

[0031] Example 2: Single-cell smears of different cell suspensions HeLa cells, mouse brain cells, and mouse liver cells were selected as cell suspensions and prepared as single-cell smears according to the method described in Example 1, followed by bright-field high-resolution scanning. The results are shown in [Figure 1]. Figure 3 A-3C.

[0032] Depend on Figure 3 It is known that when using ammonium formate solution containing bovine serum albumin for cell smears, HeLa cells, mouse brain cells, and mouse liver cells can all be uniformly distributed on ITO slides. This indicates that the single-cell smear preparation method of this application has good universality for cells of different sources and types, and can overcome the differences in size, morphology and surface characteristics of different cells, and stably achieve a single-layer, uniform smear effect.

[0033] Example 3: Unified Manifold Approximation and Projection (UMAP) Analysis Bovine serum albumin (BSA) was added to a 100 mmol / L ammonium formate solution and stirred at 300 rpm for 5 min to prepare an ammonium formate solution containing 0.02% (w / v) BSA. HeLa cell suspension was then prepared using the ammonium formate solution containing 0.02% (w / v) BSA to a HeLa cell concentration of 1 × 10⁻⁶ cells / mL. 6 A mixture of cells / mL was prepared, and the mixture was centrifuged at 1000 rpm for 4 min. The supernatant was removed to obtain HeLa resuspended cells 3. ITO slides were pre-cooled at 0℃. 10 μL of HeLa resuspended cells 3 was dropped onto an ITO slide, and then the resuspended cells were evenly spread onto the pre-cooled ITO slide using a spreader, covering an area of ​​approximately 1.5 × 1.5 cm. The ITO slide coated with HeLa resuspended cells was then baked at 30℃ for 5 min. The dried smear area was washed with 100 mmol / L ammonium formate solution to remove residual salts, and the slide was baked again at 30℃ for 5 min to obtain cell smear 3. UMAP analysis was performed on cell smear 3, and the results are shown below. Figure 4 A.

[0034] Bovine serum albumin was added to a 200 mmol / L ammonium formate solution and stirred at 500 rpm for 3 min to prepare an ammonium formate solution containing 0.02% (w / v) bovine serum albumin. Using this ammonium formate solution containing 0.02% (w / v) bovine serum albumin, mouse brain cell suspensions were prepared to a concentration of 0.5 × 10⁻⁶ cells / mL. 6 A mixture of cells / mL was prepared, and the cells were centrifuged at 1000 rpm for 4 min to remove the supernatant, yielding mouse brain cells resuspended in liquid form. ITO slides were pre-cooled to 5°C. 10 μL of the mouse brain cells resuspended in liquid form was dropped onto the ITO slide, and then evenly spread onto the pre-cooled ITO slide using a spreader, covering an area of ​​approximately 1.5 × 1.5 cm. The ITO slide coated with the mouse brain cells was then baked in a 40°C slide oven for 3 min. The dried smear area was washed with 200 mmol / L ammonium formate solution to remove residual salts, and the slide was baked again in a 40°C slide oven for 3 min to obtain cell smear 4. UMAP analysis was performed on cell smear 4, and the results are shown below. Figure 4 B.

[0035] Depend on Figure 4 It can be seen that the HeLa cell smears of this application can be divided into 9 groups, and the mouse brain cell smears can be divided into 13 groups. This indicates that the single-cell smear preparation method of this application can effectively preserve the metabolic heterogeneity between cells. Different cell types show significant clustering in metabolomics characteristics, and the clustering results are consistent with the expected biological complexity of the corresponding cell lines, which confirms the reliability and applicability of this method in single-cell metabolomics analysis.

[0036] Example 4: Comparison of different single-cell smear protocols Poly-L-Lysine treatment: Take an ITO slide and add 10 μL of a 0.01% (w / v) poly-L-Lysine solution to the area to be coated. Incubate at room temperature for 10 min, then dry at 37°C to obtain a pretreated ITO slide. Prepare a HeLa cell suspension with a concentration of 1×10⁻⁶ cells using 150 mmol / L ammonium formate solution. 6 A mixture of cells / mL was prepared, and the mixture was centrifuged at 1000 rpm for 4 min. The supernatant was removed to obtain HeLa resuspended cells 4. Pre-cooled the pretreated ITO slides to 4°C, and then 10 μL of HeLa resuspended cells 4 was evenly spread onto the ITO slides, covering an area of ​​approximately 1.5 × 1.5 cm. The ITO slides coated with HeLa resuspended cells were then baked at 37°C for 5 min to obtain cell smear 5. Cell smear 5 was subjected to bright-field high-resolution scanning; the results are shown below. Figure 5 A. Cell smear 5 was then subjected to MALDI mass spectrometry to analyze the signal intensity of four fatty acid metabolites: linoleic acid, oleic acid, stearic acid, and arachidonic acid. The results are shown in [Figure 1]. Figure 6 .

[0037] Sodium carboxymethyl cellulose treatment: Sodium carboxymethyl cellulose was added to PBS solution to prepare a PBS solution containing 0.05% (w / v) sodium carboxymethyl cellulose. HeLa cell suspension was then prepared using the PBS solution containing 0.05% (w / v) sodium carboxymethyl cellulose to a HeLa cell concentration of 1×10⁻⁶ cells / mL. 6 A mixture of cells / mL was prepared, and the mixture was centrifuged at 1000 rpm for 4 min. The supernatant was removed to obtain HeLa resuspended cells 5. 10 μL of HeLa resuspended cells 5 was evenly spread onto a pre-chilled ITO slide at 4℃, covering an area of ​​approximately 1.5 × 1.5 cm. The ITO slide coated with HeLa resuspended cells was then baked at 37℃ for 4 min to evaporate the surface solvent, resulting in cell smear 6. Cell smear 6 was subjected to bright-field high-resolution scanning; the results are shown below. Figure 5 B. Cell smear 6 was then subjected to MALDI mass spectrometry to analyze the signal intensity of four fatty acid metabolites: linoleic acid, oleic acid, stearic acid, and arachidonic acid. The results are shown in [Figure 1]. Figure 6 .

[0038] Dextran treatment: Add dextran to PBS solution to prepare a PBS solution containing 0.2% (w / v) dextran. Use the PBS solution containing 0.2% (w / v) dextran to prepare a HeLa cell suspension with a HeLa cell concentration of 1×10⁻⁶ cells. 6 A mixture of cells / mL was prepared, and the mixture was centrifuged at 1000 rpm for 4 min. The supernatant was removed to obtain HeLa resuspended cells 6. 10 μL of HeLa resuspended cells 6 was evenly spread onto a pre-chilled ITO slide at 4°C, covering an area of ​​approximately 1.5 × 1.5 cm. The ITO slide coated with HeLa resuspended cells was then baked at 37°C for 4 min to evaporate the surface solvent, yielding cell smear 7. Cell smear 7 was subjected to bright-field high-resolution scanning; the results are shown below. Figure 5 C. Cell smear 7 was then subjected to MALDI mass spectrometry to analyze the signal intensity of four fatty acid metabolites: linoleic acid, oleic acid, stearic acid, and arachidonic acid. The results are shown in [Figure 1]. Figure 6 .

[0039] Treatment with ammonium formate containing bovine serum albumin: Bovine serum albumin was added to a 150 mmol / L ammonium formate solution and stirred at 400 rpm for 4 min to prepare an ammonium formate solution containing 0.02% (w / v) bovine serum albumin. HeLa cell suspension was then prepared using this 0.02% (w / v) bovine serum albumin-containing ammonium formate solution to a HeLa cell concentration of 1×10⁻⁶ cells / mL. 6A mixture of cells / mL was prepared, and the mixture was centrifuged at 1000 rpm for 4 min. The supernatant was removed to obtain HeLa resuspended cells 7. ITO slides were pre-cooled to 4°C. 10 μL of HeLa resuspended cells 7 was dropped onto an ITO slide, and then the cells were evenly spread onto the pre-cooled ITO slide using a spreader, covering an area of ​​approximately 1.5 × 1.5 cm. The ITO slide coated with the HeLa resuspended cells was then baked at 37°C for 5 min. The dried smear area was cleaned with 150 mmol / L ammonium formate solution to remove residual salts, and the slide was baked again at 37°C for 5 min to obtain cell smear 8. Cell smear 8 was subjected to bright-field high-resolution scanning. The results are shown in [Figure 8]. Figure 5 D. Cell smear 8 was then subjected to MALDI mass spectrometry to analyze the signal intensity of four fatty acid metabolites: linoleic acid, oleic acid, stearic acid, and arachidonic acid. The results are shown in [Figure 1]. Figure 6 .

[0040] Depend on Figure 5-6 It is evident that, compared with polylysine treatment, sodium carboxymethyl cellulose treatment, and dextran treatment, the use of ammonium formate solution containing bovine serum albumin in this application for cell smear preparation shows significant advantages in terms of background treatment of the slide, cell retention rate, and signal intensity of metabolites.

[0041] Example 5: Effects of different bovine serum albumin concentrations on single-cell smears Bovine serum albumin (BSA) was added to a 150 mmol / L ammonium formate solution and stirred at 400 rpm for 4 min to prepare an ammonium formate solution containing 0.01% (w / v) BSA. HeLa cell suspension was then prepared using this 0.01% (w / v) BSA solution to a HeLa cell concentration of 1 × 10⁻⁶ cells / mL. 6 A mixture of cells / mL was prepared, and the mixture was centrifuged at 1000 rpm for 4 min. The supernatant was removed to obtain HeLa resuspended cells 8. ITO slides were pre-cooled to 4°C. 10 μL of HeLa resuspended cells 8 was dropped onto an ITO slide, and then the resuspended cells were evenly spread onto the pre-cooled ITO slide using a spreader, covering an area of ​​approximately 1.5 × 1.5 cm. The slide was then baked at 37°C for 4 min. The dried smear area was washed with 150 mmol / L ammonium formate solution to remove residual salts, and the slide was baked again at 37°C for 4 min to obtain cell smear 9. Cell smear 9 was subjected to bright-field high-resolution scanning. The results are shown in [Figure 1]. Figure 7 D-7F.

[0042] Bovine serum albumin (BSA) was added to a 150 mmol / L ammonium formate solution and stirred at 400 rpm for 4 min to prepare an ammonium formate solution containing 0.02% (w / v) BSA. HeLa cell suspension was then prepared using this 0.02% (w / v) BSA solution to a HeLa cell concentration of 1 × 10⁻⁶ cells / mL. 6 A mixture of cells / mL was prepared, and the mixture was centrifuged at 1000 rpm for 4 min. The supernatant was removed to obtain HeLa resuspended cells 9. ITO slides were pre-cooled to 4°C. 10 μL of HeLa resuspended cells 9 was dropped onto an ITO slide, and then the resuspended cells were evenly spread onto the pre-cooled ITO slide using a spreader, covering an area of ​​approximately 1.5 × 1.5 cm. The slide was then baked at 37°C for 4 min. The dried smear area was washed with 150 mmol / L ammonium formate solution to remove residual salts, and the slide was baked again at 37°C for 4 min to obtain cell smear 10. Cell smear 10 was subjected to bright-field high-resolution scanning. The results are shown in [Figure 10]. Figure 7 G-7I.

[0043] Bovine serum albumin (BSA) was added to a 150 mmol / L ammonium formate solution and stirred at 400 rpm for 4 min to prepare an ammonium formate solution containing 0.05% (w / v) BSA. HeLa cell suspension was then prepared using the 0.05% BSA ammonium formate solution to a HeLa cell concentration of 1 × 10⁻⁶ cells / mL. 6 A mixture of cells / mL was prepared, and the mixture was centrifuged at 1000 rpm for 4 min. The supernatant was removed to obtain HeLa resuspended cells 10. ITO slides were pre-cooled to 4°C. 10 μL of HeLa resuspended cells 10 was dropped onto an ITO slide, and then the resuspended cells were evenly spread onto the pre-cooled ITO slide using a spreader, covering an area of ​​approximately 1.5 × 1.5 cm. The slide was then baked at 37°C for 4 min. The dried smear area was washed with 150 mmol / L ammonium formate solution to remove residual salts, and the slide was baked again at 37°C for 4 min to obtain cell smear 11. Cell smear 11 was subjected to bright-field high-resolution scanning. The results are shown in [Figure 11]. Figure 7 J-7L.

[0044] Bovine serum albumin (BSA) was added to a 150 mmol / L ammonium formate solution and stirred at 400 rpm for 4 min to prepare an ammonium formate solution containing 0.1% (w / v) BSA. HeLa cell suspension was then prepared using this 0.1% (w / v) BSA solution to achieve a HeLa cell concentration of 1 × 10⁻⁶ cells / mL. 6A mixture of cells / mL was prepared, and the cells were centrifuged at 1000 rpm for 4 min. The supernatant was removed to obtain HeLa resuspended cells 11. ITO slides were pre-cooled to 4°C. 10 μL of HeLa resuspended cells 11 was dropped onto an ITO slide, and then the resuspended cells were evenly spread onto the pre-cooled ITO slide using a spreader, covering an area of ​​approximately 1.5 × 1.5 cm. The slide was then baked at 37°C for 4 min. The dried smear area was washed with 150 mmol / L ammonium formate solution to remove residual salts, and the slide was baked again at 37°C for 4 min to obtain cell smear 12. Cell smear 12 was subjected to bright-field high-resolution scanning. The results are shown in [Figure 12]. Figure 7 M-7O.

[0045] A separate control was prepared using ammonium formate solution without added bovine serum albumin for single-cell smears. The specific method was as follows: HeLa cell suspension was prepared to a concentration of 1×102 cells using 150 mmol / L ammonium formate solution. 6 A mixture of cells / mL was prepared, then centrifuged at 1000 rpm for 4 min. The supernatant was removed to obtain HeLa resuspended cells 12. ITO slides were pre-cooled to 4°C. 10 μL of HeLa resuspended cells 12 was dropped onto an ITO slide, and then evenly spread onto the pre-cooled ITO slide using a spreader, covering an area of ​​approximately 1.5 × 1.5 cm. The slide was then baked at 37°C for 4 min. The baked-dry area was cleaned with 150 mmol / L ammonium formate solution to remove residual salts, and the slide was baked again at 37°C for 4 min to obtain cell smear 13. Cell smear 13 was subjected to bright-field high-resolution scanning. The results are shown in [Figure 13]. Figure 7 A-7C.

[0046] Depend on Figure 7 It is known that the pure ammonium formate system exhibits poor wettability on the ITO surface due to its high surface tension, resulting in a large droplet contact angle and difficulty in effective spreading. Adding 0.01% bovine serum albumin (BSA) improves the spreading of the solution, but it cannot evenly coat the entire area. Adding 0.02% BSA allows the solution to be evenly coated on the target area. Adding 0.05% BSA requires an additional round of washing to remove the BSA, which leads to a significant reduction in cell number and a cell survival rate of 50-60%. Adding 0.1% BSA also requires an additional round of washing to remove the BSA, and while the cell survival rate is reduced to 50-60%, the high concentration of BSA can also increase osmotic pressure, causing the cells to shrink.

[0047] Example 6: Effects of different coating treatments on single-cell smears Using cell smear 8 from Example 4 as the experimental group, an ITO slide without pre-cooling was set up as control group 1, and the slide was air-dried after coating as control group 2.

[0048] Control Group 1: Bovine serum albumin was added to 150 mmol / L ammonium formate solution and stirred at 400 rpm for 4 min to prepare an ammonium formate solution containing 0.02% (w / v) bovine serum albumin. HeLa cell suspension was then prepared using the ammonium formate solution containing 0.02% (w / v) bovine serum albumin to a HeLa cell concentration of 1×10⁻⁶ cells / mL. 6 A mixture of cells / mL was prepared, then centrifuged at 1000 rpm for 4 min, and the supernatant was removed to obtain HeLa resuspended cells 13. 10 μL of HeLa resuspended cells 13 was dropped onto an ITO slide. The resuspended cells were then evenly spread onto the ITO slide at room temperature using a spreader, covering an area of ​​approximately 1.5 × 1.5 cm. The ITO slide coated with HeLa resuspended cells was observed under a microscope to confirm cell state, clumping rate, and uniformity of distribution. After confirming these were correct, the slide was baked at 37°C for 4 min. The baked-dry area was then cleaned with 150 mmol / L ammonium formate solution to remove residual salts, and the slide was baked again at 37°C for 4 min to obtain cell smear 14. Cell smear 14 was subjected to bright-field high-resolution scanning; the results are shown below. Figure 8 A.

[0049] Control Group 2: Bovine serum albumin was added to 150 mmol / L ammonium formate solution and stirred at 400 rpm for 4 min to prepare an ammonium formate solution containing 0.02% (w / v) bovine serum albumin. HeLa cell suspension was then prepared using the ammonium formate solution containing 0.02% (w / v) bovine serum albumin to a HeLa cell concentration of 1×10⁻⁶ cells / mL. 6 A mixture of cells / mL was prepared, then centrifuged at 1000 rpm for 4 min, and the supernatant was removed to obtain HeLa resuspended cells 14. ITO slides were pre-cooled to 4°C, and 10 μL of HeLa resuspended cells 14 was dropped onto the ITO slide. The resuspended cells were then evenly spread onto the pre-cooled ITO slide using a spreader, covering an area of ​​approximately 1.5 × 1.5 cm. The ITO slide coated with HeLa resuspended cells was observed under a microscope to verify cell state, clumping rate, and uniformity of distribution, and then allowed to air dry at room temperature. The dried smear area was cleaned with 150 mmol / L ammonium formate solution to remove residual salts, and the slide was then baked again at 37°C for 4 min to obtain cell smear 15. Cell smear 15 was subjected to bright-field high-resolution scanning; the results are shown below. Figure 8 B.

[0050] Depend on Figure 5 D and Figure 8As shown in A, compared to cell smear 8, the cell smear on the ITO slide without pre-cooling showed acceptable cell morphology, but some areas slightly overlapped, with minor local aggregation, resulting in reduced cell distribution uniformity. In contrast, this application uses pre-cooled ITO slides, which reduces the solvent evaporation rate on the ITO slide surface and facilitates uniform cell deposition and monolayer distribution on the ITO slide surface.

[0051] Depend on Figure 8 A, Figure 8 As shown in section B, compared to cell smear 14, where resuspended cells are coated on an ITO slide and then allowed to air dry, the cells are exposed to a hypertonic environment for an extended period, leading to metabolite leakage and cell damage. This results in a decrease in cell survival rate of over 50% and an increase in cell morphology damage rate of approximately 50%. In contrast, the present application's initial baking-washing-rebaking steps allow for rapid solvent evaporation, facilitating the rapid fixation of evenly distributed cells and creating an ideal co-crystallization microenvironment for subsequent MALDI matrix spraying. This significantly promotes the in-situ extraction and ionization efficiency of metabolites.

[0052] The above are all preferred embodiments of this application, and are not intended to limit the scope of protection of this application. Therefore, all equivalent changes made in accordance with the structure, shape and principle of this application should be covered within the scope of protection of this application.

Claims

1. A method for preparing single-cell smears suitable for matrix-assisted laser desorption / ionization, characterized in that, Includes the following steps: S1. Centrifuge the cell suspension to remove the supernatant, add ammonium formate solution containing bovine serum albumin, stir well to obtain pretreatment solution; S2. Pre-cool the indium tin oxide glass slide, then coat it with a pretreatment solution, bake it initially, clean it with ammonium formate solution, bake it again, and obtain a single-cell smear.

2. The method for preparing single-cell smears suitable for matrix-assisted laser desorption / ionization according to claim 1, characterized in that, In step S1, the concentration of bovine serum albumin in the ammonium formate solution is 0.01-0.05% (w / v).

3. The method for preparing single-cell smears suitable for matrix-assisted laser desorption / ionization according to claim 2, characterized in that, In step S1, the concentration of bovine serum albumin in the ammonium formate solution is 0.02% (w / v).

4. The method for preparing single-cell smears suitable for matrix-assisted laser desorption / ionization according to claim 1, characterized in that, The concentration of ammonium formate in the ammonium formate solution is 100-200 mmol / L.

5. The method for preparing single-cell smears suitable for matrix-assisted laser desorption / ionization according to claim 1, characterized in that, In step S2, the pre-cooling temperature is 0-5℃.

6. The method for preparing single-cell smears suitable for matrix-assisted laser desorption / ionization according to claim 5, characterized in that, In step S2, the pre-cooling temperature is 4°C.

7. The method for preparing single-cell smears suitable for matrix-assisted laser desorption / ionization according to claim 1, characterized in that, In step S2, the initial baking temperature is 30-40℃, and the re-baking temperature is 30-40℃.

8. The method for preparing single-cell smears suitable for matrix-assisted laser desorption / ionization according to claim 7, characterized in that, In step S2, the initial baking temperature is 37°C, and the re-baking temperature is 37°C.

9. The method for preparing single-cell smears suitable for matrix-assisted laser desorption / ionization according to claim 1, characterized in that, The concentration of the cell suspension was 0.5 × 10⁻⁶. 6 -1.5×10 6 per mL.

10. A single-cell smear suitable for matrix-assisted laser desorption / ionization, characterized in that, The single-cell smear was prepared using the method described in any one of claims 1-9, which is suitable for matrix-assisted laser desorption / ionization.

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