Cell lysis ionization and flow cytometry-based mass spectrometry detection device based on supercritical fluid

Abstract

This invention discloses a supercritical fluid-based cell lysis ionization and flow cytometry-mass spectrometry detection device. The device includes a supercritical fluid generator, a cell introduction device, a fluid focusing device, and a mass spectrometer detector. The supercritical fluid generator is connected to the fluid focusing device to introduce supercritical fluid. The outlet of the cell introduction device is connected to the fluid focusing device to introduce a cell dispersion of the target cells into the fluid focusing device. The fluid focusing device, driven by the supercritical fluid, focuses and generates droplets containing single-cell samples. The droplets vaporize after passing through the outlet of the fluid focusing device and are then ionized. The mass spectrometer detector is located at the outlet of the fluid focusing device. This invention solves the problem that intact cells cannot be completely ruptured during traditional electrospray ionization, and that metabolite molecules within the cells cannot be completely released to form gaseous ions. The ion clusters generated by single cells enable highly sensitive detection of the transient signals of single-cell metabolites using a mass spectrometer.

Description

Technical Field

[0001] This invention relates to a supercritical fluid-based cell lysis ionization and flow cytometry mass spectrometry detection device, belonging to the field of analytical and detection instruments. Background Technology

[0002] Mass spectrometry is a powerful tool for metabolite analysis due to its label-free, multi-component, and ability to provide molecular structure information. Among all mass spectrometry ionization techniques, electrospray ionization is best suited for the analysis of small molecule metabolites, and is therefore the most commonly used ionization method for metabolite mass spectrometry detection.

[0003] Currently, single-cell metabolite mass spectrometry analysis mainly employs two methods: one is a static method that uses a glass or quartz electrospray needle to puncture cells for sampling, followed by direct detection using electrospray mass spectrometry; the other is a microfluidic channel that arranges cells in an orderly fashion, charging droplets containing single cells under a high-voltage electric field. These droplets then break up during flight or upon entering the mass spectrometer's transport tube, causing small metabolite molecules to form gaseous ions, which are then detected by mass spectrometry. The former method is suitable for comprehensive analysis of metabolites in single cells, obtaining molecular structural information through secondary fragmentation to identify the types of metabolite molecules. However, this method is cumbersome, inefficient, and highly susceptible to human error. In recent years, this method has been developing towards automation, using robotic arms to precisely control the needle for sampling followed by mass spectrometry detection. However, in principle, this method is not suitable for high-throughput analysis of large numbers of cells. The second method is theoretically more suitable for analyzing large numbers of cells, but due to the inherent resistance of the microfluidic channel, the actual cell throughput is not high, typically on the order of tens of cells per minute.

[0004] Traditional flow cytometry's liquid flow focusing cell dispersion technology offers extremely high cell throughput, but the large liquid flow rate makes it incompatible with mass spectrometry instruments. Especially in this dynamic mode, the cells themselves acquire a charge during electrospraying and do not completely rupture during ionization. This results in incomplete release of intracellular metabolite molecules to form gaseous ions, leading to a limited number of detectable components. Compared to static single-cell metabolite mass spectrometry analysis, dynamic sample introduction analysis results in insufficient extraction of metabolites from cells, with only a few high-concentration metabolites forming gaseous ions that can be detected by mass spectrometry. Therefore, the key to improving the efficiency of single-cell mass spectrometry analysis is to provide a device that enables rapid dispersion, in-situ lysis of single cells, and instantaneous vaporization and ionization of their components. Summary of the Invention

[0005] The purpose of this invention is to provide a supercritical fluid-based cell lysis ionization and flow cytometry mass spectrometry detection device, which can rapidly disperse cell suspensions into single cells and lyse them to rapidly vaporize and ionize their components. Combined with time-of-flight mass spectrometry detection technology, it enables high-throughput analysis of single-cell metabolites.

[0006] The present invention provides a supercritical fluid-based cell lysis ionization and flow cytometry mass spectrometry detection device, comprising a supercritical fluid generator, a cell introduction device, a fluid focusing device, and a mass spectrometry detector;

[0007] The supercritical fluid generator is connected to the fluid focusing device and is used to introduce supercritical fluid into the fluid focusing device.

[0008] The outlet of the cell introduction device is connected to the fluid focusing device, and is used to introduce the cell dispersion of the cells to be tested into the fluid focusing device;

[0009] The fluid focusing device is used to focus and generate microdroplets containing single-cell samples under the drive of the supercritical fluid. The microdroplets are vaporized after passing through the outlet of the fluid focusing device and then ionized.

[0010] The mass spectrometer detector is located at the outlet of the fluid focusing device.

[0011] In the aforementioned cell lysis ionization and flow cytometry mass spectrometry detection device, the supercritical fluid generator is a supercritical carbon dioxide generator used to generate supercritical carbon dioxide fluid.

[0012] In the aforementioned cell lysis ionization and flow cytometry mass spectrometry detection device, the supercritical carbon dioxide generator includes a carbon dioxide cylinder, a pressure pump, and a heating device connected in sequence, wherein the heating device is used to increase the temperature of the carbon dioxide flow path.

[0013] In the aforementioned cell lysis ionization and flow cytometry mass spectrometry detection device, the cell introduction device includes a high-pressure injection pump and a high-pressure injection cylinder connected together, wherein the high-pressure injection cylinder is used to hold the suspension of the cells to be tested.

[0014] The high-pressure injection cylinder is connected to the fluid focusing device via an injection capillary tube.

[0015] In the aforementioned cell lysis ionization and flow cytometry mass spectrometry detection device, a high-voltage DC power supply is provided at the outlet of the fluid focusing device to perform ionization, i.e., to form ions with positive / negative charges.

[0016] A heating device is installed at the outlet of the fluid focusing device to provide energy for the phase change process of the supercritical fluid and prevent frost formation.

[0017] In the aforementioned cell lysis ionization and flow cytometry mass spectrometry detection device, the mass spectrometry detector is a quadrupole mass spectrometer, a time-of-flight mass spectrometer, and / or an ion trap mass spectrometer.

[0018] When performing single-cell mass spectrometry analysis using the cell lysis ionization and flow cytometry mass spectrometry detection device of the present invention, the following steps can be followed:

[0019] 1) Supercritical carbon dioxide fluid or a modified fluid of supercritical carbon dioxide and methanol is generated using the supercritical fluid generator in the cell lysis ionization and flow cytometry mass spectrometry detection device.

[0020] 2) The cell suspension of the cells to be tested is introduced into the fluid focusing device through the cell introduction device. Driven by supercritical fluid, the suspension is focused through the orifice of the fluid focusing device and forms a conical structure at the nozzle of the fluid focusing device. A microjet is formed at the top of the conical structure. The microjet is broken into monodisperse droplets containing the cell sample to be tested outside the orifice.

[0021] 3) After the microdroplets flow out of the fluid focusing device, they vaporize, are ionized, and then enter the mass spectrometer detector to obtain the mass spectrum of the single cell to be tested.

[0022] This invention uses supercritical carbon dioxide fluid as the sheath fluid and methanol as the supercritical modifier. It employs focused flow technology to achieve single-cell dispersion, obtain ordered single cells in high throughput, and simultaneously achieve instantaneous cell lysis and full vaporization and ionization.

[0023] Supercritical carbon dioxide possesses liquid-like properties, exhibiting excellent solubility for nonpolar substances, particularly lipids that constitute cell membranes, causing cells to rupture instantly. As an extremely compressed gas, supercritical carbon dioxide also facilitates the formation of gaseous ions from cell contents upon entering atmospheric pressure after lysis, enhancing ionization efficiency. Being a gas itself, it does not introduce matrix interference. Methanol, as a modifier, exhibits good solubility for polar substances within cells, enabling the detection of cellular energy metabolites and polar small molecules, substances typically found only in conventional flow cytometry mass spectrometry.

[0024] This invention solves the problem that intact cells cannot be completely ruptured during traditional electrospray ionization, resulting in the incomplete release of intracellular metabolite molecules to form gaseous ions. Ion clusters generated by single cells can be detected with high sensitivity using mass spectrometry to detect transient signals of single-cell metabolites.

[0025] The present invention has the following advantages due to the adoption of the above technical solutions:

[0026] 1) This invention uses a focused flow method to generate dispersed cells and arrange them in an orderly manner, which can improve the transport efficiency of cell particles and has the advantage of high sample throughput; 2) Supercritical carbon dioxide fluid, as the sheath fluid, can cause cells to lyse instantly and vaporize into carbon dioxide gas immediately after being sprayed out, without matrix effect and without interfering with subsequent mass spectrometry detection; 3) This invention uses electrospray ionization to directly ionize metabolite molecules in cells, and can obtain a large amount of molecular information in single cells through a mass spectrometry detector without labeling, which can be widely used in various mass spectrometry instruments. Attached Figure Description

[0027] Figure 1 This is a schematic diagram of the structure of the cell lysis ionization and flow cytometry mass spectrometry detection device of the present invention.

[0028] Figure 2 This is a schematic diagram of the mass spectrometry analysis results of HeLa cells in a specific embodiment of the present invention, showing the change in the extraction intensity of the m / z=760 ion recorded by the mass spectrometer detector in positive mode over time.

[0029] Figure 3 This is a schematic diagram of the fingerprint spectrum of a selected single cell recorded by the mass spectrometer detector in a specific embodiment of the present invention. Detailed Implementation

[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0031] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0032] like Figure 1 As shown, the cell lysis ionization and flow cytometry mass spectrometry detection device provided by the present invention includes a supercritical carbon dioxide fluid generator 1, a cell introduction device 2, a flow focusing device 3, and a mass spectrometer detector 4.

[0033] The key to the supercritical carbon dioxide fluid generator 1 is the generation of supercritical carbon dioxide fluid and the control of its internal pressure. To reduce the complexity of the device, supercritical carbon dioxide is obtained in a pressurized and slightly heated environment: a carbon dioxide cylinder 5, a high-pressure pump 6, and a heating device are connected in sequence. To achieve a supercritical state for carbon dioxide at 33°C, a pressure of at least 8 MPa is required. The high-pressure pump 6 and a constant pressure control module are used to maintain the internal pressure, and the heating device 7 is used to increase the temperature of the carbon dioxide flow path.

[0034] The cell introduction device 2 is used for introducing cell dispersion and includes a high-pressure injection cylinder 9 and a high-pressure injection pump 8.

[0035] The fluid focusing device 3 is used to focus the cell suspension under the drive of supercritical fluid to generate microdroplets containing single-cell samples, causing the cells to lyse instantly. After being ejected from the orifice, the microdroplets vaporize, and under the action of the external high-voltage direct current device 10, the metabolite molecules within the cells form gaseous ions. The heating device 11 provides energy for the phase transition process generated by the supercritical fluid and prevents frost formation. Specifically, the outlet of the cell introduction device 2 is connected to the fluid focusing device 3 through the sample inlet capillary 12. The high-voltage direct current device 10 can be directly connected to the cell introduction device 2 or added after the fluid focusing device 3. The high-voltage direct current power supply 10 is used to generate electrospray by applying a high voltage to the single-cell particle beam. The outlet of the fluid focusing device 3 is connected to the mass spectrometer detector 4, which is used to obtain the spectrum of single-cell ions by mass spectrometry.

[0036] In a preferred embodiment, the mass spectrometer detector 4 is a quadrupole mass spectrometer, a time-of-flight mass spectrometer, and / or an ion trap mass spectrometer.

[0037] In a preferred embodiment, the high-pressure sample injection cylinder 9 in the cell introduction device 2 is made of stainless steel and can withstand a pressure of 20 MPa.

[0038] In a preferred embodiment, the fluid focusing device 3 has a gas interior and a liquid exterior, with a certain liquid flow rate Q. l and gas flow rate Q g Driven by the fluid focusing device 3, the fluid is focused through the small hole and forms a stable conical structure at the tube opening. A microjet is formed at the top of the conical structure. The microjet breaks into monodisperse microdroplets containing the cell sample to be tested at a certain distance outside the small hole. The fluid focusing device 3 is a device disclosed in the prior art, and its specific structure will not be described in detail here.

[0039] In a preferred embodiment, the voltage of the electrospray ion source of the high-voltage direct current device 10 is 2 to 3 kV, and it is located behind the fluid focusing device 3 to generate charged ions from cell contents.

[0040] When performing flow cytometry mass spectrometry analysis using the cell lysis ionization and flow cytometry mass spectrometry detection device of the present invention, the following steps can be followed:

[0041] 1) Carbon dioxide enters the pressure pump 6 through the carbon dioxide cylinder 5, mixes with methanol to form a carbon dioxide / methanol liquid with a pressure higher than 8MPa, and after passing through the heating device 7, it reaches the specified temperature to form supercritical carbon dioxide, which then flows into the flow focusing device 3.

[0042] 2) Disperse the cells to be tested in a buffer solution to obtain a cell dispersion of the cells to be tested, and add it to the high-pressure injection cylinder 9.

[0043] 3) The high-pressure injection pump 8 applies a pressure higher than the supercritical carbon dioxide pressure, pushing the cell dispersion to be tested into the injection capillary 12 with an inner diameter of 30-50 μm and into the fluid focusing device 3. Under the impetus of the supercritical carbon dioxide fluid, the dispersion is focused through the small hole of the fluid focusing device 3 and forms a stable conical structure at the tube opening of the fluid focusing device 3. A microjet is formed at the top of the conical structure. The microjet breaks into monodisperse droplets containing the cell sample to be tested at a distance of 0.5-2.0 cm outside the small hole.

[0044] 4) The generated droplets containing the single-cell sample to be tested form ions with positive / negative charges under the combined action of the heating device 11 and the high-voltage direct current device 10, wherein the high-voltage ionization device applies a high-voltage direct current of 2k to 3kV (positive high voltage / volt high voltage).

[0045] 6) The formed ions enter the mass spectrometer detector 4, and the mass spectrum of the single cell to be tested is obtained by high-resolution mass spectrometry detection.

[0046] like Figure 2 The diagram shows the results of mass spectrometry analysis of the HeLa cells to be tested. It also shows the change in the extraction intensity of the m / z=760 ion recorded by the mass spectrometer detector over time. One pulse signal represents the successful detection of one HeLa single cell.

[0047] like Figure 3 As shown, Figure 2 A fingerprint pattern of a selected HeLa single cell was used to retrieve and confirm the metabolite information of the HeLa single cell by searching a mass spectrometry database.

Claims

1. A supercritical fluid-based cell lysis ionization and flow cytometry mass spectrometry detection device, comprising a supercritical fluid generator, a cell introduction device, a fluid focusing device, and a mass spectrometry detector; The supercritical fluid generator is a supercritical carbon dioxide generator. The supercritical carbon dioxide generator includes a carbon dioxide cylinder, a pressure pump, and a heating device connected in sequence; the supercritical fluid generator is connected to the fluid focusing device and is used to introduce supercritical fluid into the fluid focusing device. The outlet of the cell introduction device is connected to the fluid focusing device, and is used to introduce the cell dispersion of the cells to be tested into the fluid focusing device; The cell introduction device includes a connected high-pressure injection pump and a high-pressure injection cylinder. The high-pressure injection cylinder is connected to the fluid focusing device via an injection capillary tube. The fluid focusing device is used to focus and generate microdroplets containing single-cell samples under the drive of the supercritical fluid. The microdroplets are vaporized after passing through the outlet of the fluid focusing device and then ionized. The mass spectrometer detector is located at the outlet of the fluid focusing device; A high-voltage DC power supply is installed at the outlet of the fluid focusing device for ionization.

2. The cell lysis ionization and flow cytometry mass spectrometry detection device according to claim 1, characterized in that: A heating device is installed at the outlet of the fluid focusing device.

3. The cell lysis ionization and flow cytometry mass spectrometry detection device according to claim 1 or 2, characterized in that: The mass spectrometer detector is a quadrupole mass spectrometer, a time-of-flight mass spectrometer, or an ion trap mass spectrometer.

4. A single-cell mass spectrometry analysis method, comprising the following steps: 1) Using the supercritical fluid generator in the cell lysis ionization and flow cytometry mass spectrometry detection device according to any one of claims 1-3 to generate supercritical carbon dioxide fluid or a modified fluid of supercritical carbon dioxide and methanol mixture; 2) The cell suspension of the cells to be tested is introduced into the fluid focusing device through the cell introduction device. Driven by supercritical fluid, the suspension is focused through the orifice of the fluid focusing device and forms a conical structure at the nozzle of the fluid focusing device. A microjet is formed at the top of the conical structure. The microjet is broken into monodisperse droplets containing the cell sample to be tested outside the orifice. 3) After the microdroplets flow out of the fluid focusing device, they vaporize, are ionized, and then enter the mass spectrometer detector to obtain the mass spectrum of the single cell to be tested.

5. The application of the cell lysis ionization and flow cytometry mass spectrometry detection device according to any one of claims 1-3 in single-cell analysis.

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