Living cell in-situ detection method based on glass electrospray ionization mass spectrometry

Abstract

The invention belongs to the field of analytical chemistry, and particularly relates to a living cell in-situ detection method based on glass electrospray ionization mass spectrometry. The system provided by the invention is composed of a glass slide part for culturing cells, a glass electrospray part and a mass spectrum online detection part. The invention provides a novel living cell in-situ detection method based on glass spray ionization mass spectrometry aiming at the vacancy in the single cell in-situ rapid detection direction. The method not only can realize in-situ detection of cells, but also can monitor and analyze change of substances in the cells through atmospheric pressure mass spectrometry; the method fills the technical blank in the field; the method provided by the invention can realize real-time, continuous and online analysis of the sample without pretreatment of the sample, and plays an important role in the aspects of substance structure analysis and mechanism exploration. Therefore, when the probe is used for in-situ detection of living cells, substances in the cells can be better explored, and practical application of the probe is promoted.

Description

Technical Field

[0001] The patent of this invention belongs to the field of analytical chemistry, and specifically relates to an in situ detection method of living cells based on glass electrospray ionization mass spectrometry. Background Art

[0002] In recent years, with the development of society, the incidence of cancer is increasing rapidly around the world due to population growth and aging, environmental pollution, unhealthy lifestyles, etc. Therefore, studying the components in cancer cells and their inhibition methods has become one of the key issues in the development of human health. The emergence of single-cell analysis technology in recent years has provided a powerful tool to solve this difficulty and advance cancer research. Single-cell analysis has more and more advantages in elucidating biological variability and understanding susceptibility to different diseases. In particular, real-time detection of living cells can accurately and quickly evaluate cell status, which ultimately helps to develop more effective medical interventions.

[0003] In previous biological experiments, cells usually grow adherently on the surface of culture dishes, and cell suspension treatment is required for rapid detection. Compared with traditional cell detection technology, surface-based in situ detection can quickly and easily detect living cells, and is an ideal method for single-cell detection. At present, the most important surface ionization method for mass spectrometry detection is paper spray ionization (PSI), which is electrospray ionization (ESI) of the sample from the tip of paper under high voltage. Although this method has the advantages of less pretreatment and simple structure, it still has problems such as strong adsorption and unpredictable contamination or damage to living cells, so it cannot be used for living cell detection. Summary of the invention

[0004] In view of the problems that the current live cell detection pre-processing is complicated and easy to cause pollution and damage to cells, the present invention provides a live cell in situ detection method based on glass spray ionization mass spectrometry. The method places a glass slide into a culture dish containing cells, so that the cells grow on the glass slide. The intracellular substances are detected by atmospheric pressure mass spectrometry, the tip of the glass slide is close to the mass spectrometer detector and connected to the mass spectrometer high voltage to ionize the cells, so as to accurately obtain the structural information of the intracellular molecules and further explore the changes of intracellular substances.

[0005] The technical solution adopted by the present invention to achieve the above-mentioned purpose is: The present invention provides a living cell in situ detection method based on glass spray ionization mass spectrometry, which is composed of a glass slide part for culturing cells, a glass electrospray part and a mass spectrometry online detection part: The glass slide portion carrying the cells: the cells grow evenly on the glass slide; a mixed solvent is dripped on it, and a spray is generated at the tip of the glass slide under the action of high voltage; The droplet electrospray part: the tip of the glass sheet is tilted close to the mass spectrometer port, and the external high voltage from the mass spectrometer is connected to the rear end of the glass sheet, so that the liquid is transformed into spray and enters the mass spectrometer detector; The mass spectrometer online detection part: the electrospray generated at the tip of the glass sheet is combined with the mass spectrometer detector to ionize the substances in the living cells and enter the mass spectrometer for detection.

[0006] Preferably, the specific culturing method of the glass slide carrying cells is: placing the ultra-thin glass slide into a culture medium containing cells, and culturing in a humidified environment of 37°C and 5% CO2 to allow the cells to grow evenly attached to the wall.

[0007] Preferably, the culture medium is DMEM culture medium; 10% FBS, 100 μ / mL penicillin and 100 mg / mL streptomycin are added to the culture medium.

[0008] Preferably, the ultra-thin glass slide used in the present invention is an equilateral triangle with a vertex angle of 60°, a side length of 20 mm, and a thickness of 0.2±0.02 mm.

[0009] Preferably, the mixed solvent is a methanol-water solution; in the methanol-water solution, the volume ratio of methanol to water is 1:1.

[0010] Preferably, the distance between the tip of the glass sheet and the mass spectrometer detector is 5-10 mm, the inclination angle is between 10-30°, and the mass spectrometer high voltage is 1.0-4.0 kV; preferably, the high voltage is 3.5 kV.

[0011] Preferably, the detection conditions of the mass spectrometry are: m / z 80~500, full scan test in negative ion mode; mass spectrometry injection capillary temperature (Capilary Temp) is 275 °C; capillary voltage (Capilary Voltage) is -26 V, tube lens compensation voltage (Tube lens) is -67 V; ion injection time is up to 10 ms, and cell metabolites and standard samples are confirmed by collision induced dissociation (CID).

[0012] The present invention uses a glass sheet as a substrate for living cell attachment and culture, which has less adsorption of samples and is conducive to real-time monitoring of electrochemical reactions when ESI is performed on the top of the triangular glass. Using glass as a substrate can avoid problems such as adverse adsorption, contamination or damage to living cells. Under the action of external high voltage electricity, the solution containing cells can easily form an electrospray, thereby quickly entering the mass spectrometry detection to achieve the detection of intracellular substances. In addition, the device can effectively and controllably avoid problems such as adsorption and damage of the matrix to the cells, and better achieve in situ detection of cells.

[0013] The method provided by the present invention, atmospheric pressure mass spectrometry can directly sample at atmospheric pressure, has the characteristics of high resolution, high sensitivity, high throughput, easy operation, etc., and can realize real-time, continuous, and online analysis of samples without sample pretreatment, playing an important role in material structure analysis and mechanism exploration. Therefore, using it for in situ detection of living cells can better explore intracellular substances and promote its practical application.

[0014] The beneficial effects of the present invention are: (1) This invention addresses the lack of rapid in situ detection of single cells and provides a new method for in situ detection of living cells based on glass spray ionization mass spectrometry. This method can not only realize in situ detection of cells, but also monitor and analyze changes in intracellular substances through atmospheric pressure mass spectrometry; this method fills the technical gap in this field; (2) The method provided by the present invention can realize real-time, continuous and online analysis of samples without pre-treatment of the samples, which plays an important role in the analysis of material structure and exploration of mechanism. Therefore, using it for in situ detection of living cells can better explore intracellular substances and promote its practical application.

[0015] (3) After the detection, the cells remaining on the glass slide can be recovered and cultured, thereby improving the consistency of the detection. This method can overcome the damage to cells caused by traditional methods, and can be used to quickly detect living cells in a simple and effective manner, and has the potential to monitor changes in cell metabolites. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Schematic diagram of the glass slide part carrying cells, the droplet electrospray part and the mass spectrometer online detection part; Figure 2 The mass spectra of the standard substances GSH, LA and PC detected by the method provided in Example 1; Figure 3 Mass spectra of living cells detected by the method provided in Example 1; Figure 4 Mass spectrogram of detecting reactive oxygen species in cells using the method provided in Example 1 Figure 5 Image of cell migration on the tip of a glass slide; Figure 6 Cell activity images before and after in situ glass electrospraying. DETAILED DESCRIPTION

[0017] The technical solution of the present invention is further explained and illustrated by means of specific embodiments below.

[0018] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0019] 1. Materials and reagents Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0020] The water used in the experiment was ultrapure water, which was treated by the Milli-Q ultrapure water purification system.

[0021] The instrument used in the following examples is a LTQ linear ion trap mass spectrometer (Thermo Fisher Scientific, USA).

[0022] The equilateral triangle glass slide with a vertex angle of 60°, a side length of 20 mm, and a thickness of 0.2 mm was customized by Xinhe Technology Development Co., Ltd.

[0023] During the entire experiment, the spray voltage was 3.5 kV, the vacuum interface temperature was 275 °C, and the remaining experimental parameters were automatically optimized by the software.

[0024] Example 1 Human cervical adenocarcinoma epithelial cells (HeLa) and human non-small cell lung cancer cells (A549 cells) were used as detection objects for method validation.

[0025] (1) Construction of in-situ glass electrospray device A custom equilateral triangle glass slide (vertex angle 60°, side length 20 mm, thickness 0.2 mm) was placed at the bottom of the culture dish, and the cell suspension was added to the culture dish, and then the prepared culture medium (DMEM culture medium supplemented with 10% FBS, 100 μ / mL penicillin and 100 mg / mL streptomycin) was added to culture the cells. It was placed in a 37°C, CO2 content 5% incubator for 24 h to allow the cells to adhere to the glass slide. The cell density of one glass slide was about 6×10 5 . The triangular glass slide with living cells attached can be directly used for in situ glass electrospray mass spectrometry detection. As shown in Figure 1, about 20 µL of solvent (methanol: water = 1:1, V / V) was added to the glass slide with cells attached. Then, high voltage (3.5 kV) was directly applied to the surface of the glass slide soaked in solvent to initiate the electrical migration of living cells to the tip of the glass slide. Thereafter, under the action of the high voltage electric field between the glass slide and the mass spectrometer detector, the cells on the top of the glass were released and detected by in situ glass electrospray mass spectrometry. The top of the glass was 5 mm away from the mass spectrometer inlet.

[0026] It is worth noting that when pure water is used as the solvent, there is no signal under the action of high voltage.

[0027] (2) Performance of in-situ glass electrospray device In order to test the performance of the device, some standard samples were used as models for testing. During the test, 1 mM glutathione (GSH), linoleic acid (LA) and phosphatidylcholine (PC) sample solutions were directly dropped on the surface of the glass slide for detection. Figure 2 As shown, [GSH-H] - (m / z 306), [LA-H] - (m / z 279) and PC + The characteristic ion pairs of (m / z 758) were detected, and their structures were further confirmed by CID experiments. This shows that the method based on in-situ glass electrospray provided by the present invention can quickly, simply and effectively detect samples on glass slides.

[0028] To test the performance of rapid direct detection of live cells cultured on glass slides, HeLa cells and human non-small cell lung cancer cells (A549 cells) were selected as models. Since both cell lines contain lipids, the corresponding lipids in both cells were detected. In order to collect structural information on lipid-related metabolites in living cells, negative ion mode was used to detect species in the m / z 80-500 range. As shown in Figure 3a, lactate ([LA-H] - , m / z 89) and stearic acid ([SA-H] - , m / z 283) and other important ions. In A549 cells, other metabolites were observed, including linoleic acid ([LA-H] - , m / z 279) and hexose ([Hex+Cl] - , m / z 215) (Figure 3b). In addition, [4-HNE-H] at m / z 155 was also recorded in A549 cells. - This is consistent with the characteristics of overexpression of unsaturated fatty acids in A549 cells.

[0029] To further evaluate the ability to monitor structural changes in living cells, this method was used to monitor reactive oxygen species (ROS) in HeLa cells. The structural changes of the probe 2',7'-dichlorodihydrofluorescein (DCFH-DA) after being taken up by cells reflect the intracellular ROS content. In the experiment, 50 µL DCFH-DA (10 mM) was added to the culture dish containing the glass slide and incubated for 30 minutes.

[0030] like Figure 4 As shown, in HeLa cells cultured with DCFH-DA, [DCFH-DA +Na-2H] -at m / z 507 and [DCFH- CO2+Na-2H] - The mass spectrometry signal at m / z 379 indicates that DCFH-DA can enter cells and be hydrolyzed to DCFH by cellular hydrolases. After adding ROS-inducing agents to cells, [DCF-H] - The oxidation product at m / z 399 confirmed the generation of ROS. Therefore, the structural changes of DCFH-DA can be monitored in situ by glass electrospray mass spectrometry to reflect the formation of ROS in cells.

[0031] (3) Migration of living cells on glass slides Since living cells grow on the surface of glass slides, the migration of living cells to the tip of the triangular glass slide becomes a prerequisite for in situ glass electrospraying. In order to study the migration of cells under high pressure, human cervical cancer cells (HeLa cells) were selected as a model and cultured on the surface of glass slides. The magnified image of a single cell on the triangular tip of the glass slide was recorded by optical microscopy.

[0032] As shown in Figure 5a, without turning on the high voltage (0 s), cells 1 to 3 adhered can be well observed. However, at 0.3 s after turning on the high voltage (Figure 5b), the cells began to migrate toward the top of the glass slide, especially cells 1 and 2. Thereafter, at 0.8 s (Figure 5c), the cells moved further to the vicinity of the triangular tip. Finally, at 1.6 s (Figure 5d), cells 1 and 2 were ejected and no records were recorded on the glass slide. In addition, cell 3 also migrated to the top of the glass slide and is expected to be ejected soon. Therefore, under the high pressure and solvent system, the living cells adhered to the glass slide can migrate toward the triangular tip of the slide. This facilitates the subsequent in situ glass electrospray detection of living cells without any pretreatment.

[0033] (4) Effects of in situ glass electrospray on living cells Furthermore, the effect of electrospray on the biological activity of living cells remaining on the glass slide was investigated. In order to detect the biological activity of the cells, the HeLa cells attached to the glass slide were placed back into the incubator under the same conditions as above and cultured for 24 h, and then detected by confocal imaging.

[0034] As shown in Figure 6a, before the experiment, a high density of HeLa cells adhered to the glass slide. After in situ glass electrospraying (Figure 6b), most of the cells were ejected, and only a small number of cells remained on the glass slide. Then, the remaining cells on the glass slide were cultured in culture medium for 24 hours, and then cell images were collected. As shown in Figure 6c, the density of HeLa cells on the glass slide increased significantly, indicating that the biological activity of the cells was maintained after in situ glass electrospraying. This proves that this method can provide potential monitoring possibilities for detecting multiple cells in a biological process.

[0035] result The rapid detection of living cells using electrospray was studied, and a glass slide was introduced into the electrospray system to build an in-situ glass electrospray device. A new method for in-situ analysis of living cells using glass slide droplet spray ionization was developed. Under high voltage, the cells detached from the glass slide and entered the solution, forming a droplet spray, thereby achieving mass spectrometry detection. After the experiment, the cells remaining on the glass slide can be recovered and continued to be cultured, thereby improving the consistency of detection. This method can overcome the damage to cells caused by traditional methods, and can simply and effectively perform rapid detection of living cells, and has the potential to monitor changes in cellular metabolites.

Claims

1. A method for in situ detection of living cells based on glass spray ionization mass spectrometry, characterized in that: It consists of a glass slide part for culturing cells, a glass electrospray part and a mass spectrometer online detection part: The glass slide portion carrying the cells: the cells grow evenly on the glass slide; a mixed solvent is dripped on it, and a spray is generated at the tip of the glass slide under the action of high voltage; The droplet electrospray part: the tip of the glass sheet is tilted close to the mass spectrometer port, and the external high voltage from the mass spectrometer is connected to the rear end of the glass sheet, so that the liquid is transformed into spray and enters the mass spectrometer detector; The mass spectrometer online detection part: the electrospray generated at the tip of the glass sheet is combined with the mass spectrometer detector to ionize the substances in the living cells and enter the mass spectrometer for detection.

2. The method for in situ detection of living cells according to claim 1, characterized in that: The specific culturing method of the glass slide carrying cells is: placing the ultra-thin glass slide into a culture medium containing cells, and culturing in a humidified environment of 37°C and 5% CO2 to allow the cells to grow evenly attached to the wall.

3. The method for in situ detection of living cells according to claim 2, characterized in that: The culture medium is DMEM culture medium; 10% FBS, 100 μ / mL penicillin and 100 mg / mL streptomycin are added to the culture medium.

4. The method for in situ detection of living cells according to any one of claims 1 to 3, characterized in that: The ultra-thin glass slide is an equilateral triangle with a vertex angle of 60°, a side length of 20 mm, and a thickness of 0.2±0.02 mm.

5. The method for in situ detection of living cells according to any one of claims 1 to 4, characterized in that: The mixed solvent is a methanol-water solution; in the methanol-water solution, the volume ratio of methanol to water is 1:

1.

6. The method for in situ detection of living cells according to any one of claims 1 to 5, characterized in that: The distance between the tip of the glass sheet and the mass spectrometer detector is 5-10 mm, the tilt angle is between 10-30°, and the mass spectrometer high voltage is 1.0-4.0 kV; preferably, the high voltage is 3.5 kV.

7. The method for in situ detection of living cells according to any one of claims 1 to 6, characterized in that: The detection conditions of the mass spectrometer are as follows: m / z 80~500, full scan test in negative ion mode; the mass spectrometer injection capillary temperature (CapilaryTemp) is 275°C; the capillary voltage (Capilary Voltage) is -26 V, and the tube lens compensation voltage (Tube lens) is -67 V; the maximum ion injection time is 10 ms, and cell metabolites and standard samples are confirmed by collision induced dissociation (CID).

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